KR102205804B1 - Systems and methods for evacuating an array at the point of use - Google Patents

Abstract

The present invention provides a system, method and member for evacuating and filling an array at the point of use.

Description

Systems and methods for evacuating an array at the point of use

The present invention generally relates to a device for evacuating and filling a chamber at the point of use in a closed system (eg, an array of reaction wells or reagent blisters).

Infectious diseases in the United States, Canada and Western Europe account for about 7% of human mortality, while infectious diseases in developing countries account for about 40% of human mortality. Infectious diseases cause a variety of clinical symptoms. Among the commonly obvious symptoms include fever, pneumonia, meningitis, diarrhea and diarrhea blood. Physical symptoms suggest some pathogens and eliminate others as etiology, but various potential causative factors remain, so a clear diagnosis requires various analyzes. Traditional microbiological techniques for diagnosing pathogens can take days or weeks, delaying proper treatment.

Recently, polymerase chain reaction (PCR) has become a method of choice for rapid diagnosis of infectious agents. PCR can be a fast, sensitive, and specific tool for diagnosing infectious diseases. However, for a variety of possible causative organisms or viruses, and low levels of organisms or viruses present in some pathological samples, using PCR as a primary diagnostic tool can be a challenge. It may be impractical to use a very large panel of PCR analysis, one for each probable causative organism or virus that is most likely to be negative. The problem can be exacerbated when the pathogen nucleic acid is in very low concentrations, requiring a large number of samples to collect a suitable reaction template. In some cases, there may be inadequate samples to analyze all possible prevalence factors. The solution is to "perform multiplex PCR, where samples are analyzed for multiple targets simultaneously in a single reaction. Multiplex PCR has proven useful in some systems, but has a high degree of robustness against complex reactions. (Robustness) and difficult to clearly analyze a large number of products To solve this problem, the analysis will have to be divided into a plurality of secondary PCRs afterwards, if the second reaction is overlapped in the first product, the robustness is Increased robustness Closed systems such as FilmArray® (BioFire Diagnostics, LLC, Salt Lake City, UT) can reduce the risk of contamination by reducing manipulation.

Various analytical systems and methods include multi-well arrays as a means of performing analyzes on large volumes of samples. Typically each well as a sample container is to provide a standalone assay. Thus, the well and the analyte material within the well are generally designed to be separated from each other; There is no cross-talk with each other. For this reason, it can be difficult to mount each well of the array, especially in a closed system. For example, in a closed array system, a plurality of wells may be individually mounted by separate fluid channels extending into each well to reduce cross contamination with each other. However, it is difficult to fill such a channel so that bubbles are not generated, and bubbles in the wells may cause problems such as reducing the amount of samples in some wells, and detection problems may occur. In addition, such a system has a high manufacturing cost and is difficult to manufacture. In addition, the use of the separated fluid channel may cause differences in materials for each well, resulting in an analysis result.

For another system, all or part of the array can be immersed in the material at the same time, and restrictive openings to each well can be used to reduce contamination between wells. For example, the array may have a film cover that seals the wells, and perforations may be disposed on the film of each well. This has been disclosed in US Patent No. 8,895,295. The fluid material may be dispersed on the surface of the array cover film, through which a part of the fluid material may be introduced into each well through each perforation. The perforation can be configured to limit fluid outflow from the well. For example, it can be configured so that no force is applied.

In various systems, a pressure differential can be used to mount the well to the fluid. For example, positive pressure may be used to force the sample through the pathway of the fluid, if applicable, through the perforation, and into the well. However, if the pressure in the system is high, the application of positive pressure can damage the structural features associated with the array. Accordingly, some existing systems that use positive pressure and separate fluid channels to fill each well use an exhaust (vent) or overflow system (e.g. "Out" channel) However, the method requires an accurate measurement of the amount of fluid applied to the system, which can result in different fill amounts from well to well or from sample container to sample container. In addition, the exhaust channels can unintentionally introduce contaminants into the array system, which can damage the device and lead to erroneous results. In addition, while some air located in the fluid pathway is discharged by the incoming fluid, air bubbles may be trapped in the pathway to affect sample analysis, and while the air bubbles are heating As it expands, it is possible to increase the cross-talk during the analysis of the thermal cycle.

As an alternative to this, negative pressure or vacuum can be used to draw the fluid material into each well. Existing systems employing separate fluid channels to fill into each well can evacuate air from the wells and fluid channels by creating a vacuum through the fluid sample. Thus, when the vacuum is released, fluid material is sucked into each well through the fluid channel. However, the vacuum level (or pressure difference) is limited by the partial pressure of the fluid sample. For example, the partial vapor pressure of water is about 25-32 mba at room temperature (ie, 20-25° C.) and 1 atmosphere, and the partial vapor pressure of water may increase as the vacuum progresses. Thus, in the presence of water or other fluid, only a limited amount of air can be removed from each well.

In the case of a conventional array flooding system, a vacuum may be applied or formed when provided. For example, the array may be sealed in the evacuation chamber so that the array is assembled under vacuum (or reduced pressure) conditions. The array system can then be packaged, stored and moved while the vacuum application is maintained during manufacture. However, such systems require materials and/or packaging that can maintain an adequate level of vacuum until use, which can add significant cost to the product.

Thus, the need for products, methods and systems for in-situ evacuation and filling of chambers in closed systems (e.g., arrays of reaction wells or reagent blisters), in real time of chambers in closed systems There is a need for a device configured for in-situ exhaust, and for a method for manufacturing a reaction vessel configured for in-situ exhaust.

U.S. Patent No. 8,895,295

An object of one aspect of the present invention is to a system and method for evacuating an array at the point of use.

To achieve the above object,

An embodiment of the present invention is one of the aforementioned or other problems with a recently new system and method for exhausting at the point of use, preferably a system and method for sequentially filling one or more chamber types, particularly a closed atmosphere chamber type. You can solve the above. For example, an embodiment may include an array assembly that can be evacuated at the point of use and/or upon request in real time (ie, in performing an analysis method). Thus, preferably, the vacuum is filled to fill with liquid from a few seconds to minutes (i.e. long enough to form a vacuum and reseal the system, preferably a vented chamber (e.g., a well array). It can be held for a long enough time to open the seal). Thus, an almost completely formed vacuum can be used to suck fluid into the array assembly when opening the fill seal.

According to one embodiment, the vacuum is not limited to the vapor pressure of the liquid (eg, water) because the vacuum is not formed through the liquid sample. The level of depressurization of a strong vacuum can completely exhaust air from the array assembly, thus minimizing residual air remaining on the array assembly.

For example, when the vacuum is released, the strong vacuum can constantly suck the entire fluid into each of the wells. By completely exhausting the air in the array assembly, air bubbles in the sucked fluid can be reduced, initialized, and preferably eliminated compared to conventional systems. In addition, the array assembly may have a shape (eg, size and structure) that does not require an exhaust chamber for applying a vacuum. Instead, in one embodiment, a piston pump or other vacuum member may be used to form a state closer to a complete vacuum state within the array assembly.

The array assembly according to an embodiment may have a plurality of wells in an array form, a vacuum channel in fluid communication with a plurality of wells and a vacuum port, and an array filling channel in fluid communication with a plurality of wells and a fluid reservoir. The fluid channel and the vacuum channel may be co-aligned, co-extensive, and in some embodiments the same. The openable fill seal may be disposed in a fill channel or in a fluid channel disposed between the fill channel and the fluid reservoir. According to an embodiment, the fill seal may be or include an openable seal (eg, a burst seal or a peel seal), such as a seal formed by folding a fill channel or a fluid channel. . A vacuum member can be connected to the vacuum port, and vacuum can be applied to the array assembly (ie, vacuum channels, multiple wells and fill channels).

The vacuum seal may preferably be placed on a vacuum channel (eg, a vacuum channel disposed between the vacuum port and a plurality of wells) to maintain a vacuum in the well. An openable fill seal, preferably an openable fill seal that can be located between the array and another blister (e.g., fluid reservoir), allows the array assembly to be in a vacuum state. It can prevent exhaust of the fluid reservoir while in. When the fill seal is open, the fluid in the reservoir can be sucked into a plurality of wells through the fluid channel and the array fill channel. In a plurality of wells, the fluid may be mixed with one or more reagents.

According to an embodiment, the well sealing part may be formed in at least some of the plurality of wells. For example, a segment of the array fill channel may extend through a row of wells in the array. Accordingly, a portion of the filling channel may communicate with wells of the row, and may not be directly communicated with wells of adjacent or other rows. Each well may be sealed or blocked from all other wells by forming a well sealing portion such as thermal welding between the card layer and the second film layer, a row of the well, and a portion of the filling channel. In this way, cross-talk between array wells can be minimized or eliminated.

According to an embodiment, the openable fill seal may be formed in the fluid reservoir and the fluid channel in fluid communication with the fluid channel. Since the array assembly does not need to be assembled under a vacuum such as a vacuum chamber, the array assembly does not need to be packaged, stored and transported in a vacuum packaging container. The present invention can significantly save labor and material costs, and can more easily provide consumers with an analytical technique performed with the array assembly. Further, embodiments of the present invention may have a smaller material space, since there is no need to use a strong, vacuum-sealed packaging container.

According to an embodiment, the array assembly includes a card layer including a plurality of wells therein, a first layer coupled to a first surface of the card layer, and/or a second layer coupled to a first surface of the card layer. It may include a film layer. The first film layer may seal the first end of each of the plurality of wells. The second film layer may at least partially seal the second ends of the plurality of wells. The first film layer and the second film layer may be in the form of a pouch in which a card layer is disposed. For example, the card layer may be laminated between the first film layer and the second film layer by adhesive or thermoforming.

In another embodiment, the card layer, the first film layer, and/or the second film layer is used to form the vacuum channel and/or the array filling channel, and/or the vacuum channel and/or the array filling channel. Forming the can be formed to assist. For example, in at least one embodiment, the vacuum channel and/or the filling channel may be formed in the card layer, or, for example, may affect a part of the surface of the card layer. According to an embodiment, the vacuum channel and/or the filling channel may be formed on the second film layer or a portion of the surface of the second film layer.

The second film layer may be combined with the third film layer, through which the vacuum channel and/or the filling channel may be formed between the second film layer and the third film layer. The third film layer may have a plurality of perforations. The perforations may be aligned and/or may be in fluid communication with a plurality of wells, the vacuum channels, and/or fill channels.

The array assembly may be manufactured by bonding the first film layer to the first surface of the card layer and bonding the second film layer to the second surface of the card layer to form a vacuum channel and an array filling channel. According to an embodiment, the first film layer and the second film layer may be combined with each other to form a card layer pouch. Bonding the film layers together can form fluid channels, fluid reservoirs, and/or other components of the sample container.

The card layer (for example, a card layer having a concave conduit formed therein) may be selectively prepared as a reagent in a plurality of wells, and the inside of the pouch through an opening formed by separation of the first and second film layers. Can be injected with. The first and second film layers may be bonded to opposite surfaces of the card layer, and by heat welding the opening of the pouch, the pouch may be sealed inside the card layer.

One aspect of the present invention,

A first layer and at least one second layer;

A third layer disposed between the first layer and the second layer and having a plurality of wells therein;

A vacuum channel in fluid communication with a plurality of wells and vacuum ports; And

An array assembly including a plurality of wells and an array filling channel in fluid communication with the fluid source may be provided.

The first layer and the second layer may be a film layer.

The third layer may include a card layer in which a plurality of wells are formed.

The first layer is bonded to the first surface of the card layer to seal the first end of each of the plurality of wells, and the second layer is bonded to the second surface of the card layer to seal the plurality of wells. (well) Each second stage can be sealed.

The vacuum port may include an opening in at least one of the first layer and the second layer.

The array assembly includes a concave conduit in the card layer, and at least a portion of at least one of the vacuum channel and the array filling channel is formed between the second film layer and the concave conduit of the card layer,

The concave conduit of the card layer is a manifold that fluidly communicates a plurality of wells to the vacuum port by at least two paths, and fluidly communicates each well to the fluid power source by at least two paths. It can have a fold structure.

At least a portion of the vacuum channel may be disposed in at least a portion of the array filling channel and/or may be co-localized.

An open seal may be further included in the array filling channel between the plurality of wells and the fluid source.

The plurality of wells are disposed in each of the plurality of rows,

The array fill channel includes a connection channel in fluid communication with the fluid source and the manifold channel assembly,

The manifold channel assembly,

A plurality of branch channels in fluid communication with each of the plurality of wells and extending along each of the plurality of rows; And a first main channel extending along the first end of the plurality of rows,

The plurality of branch channels may extend from the first main channel, and the array filling channel may be in fluid communication with the first main channel.

The manifold channel assembly further includes a second main channel in fluid communication with the plurality of branch channels, the second main channel extending along a second end of the plurality of rows, and the vacuum port is the second In communication with the main channel, each well can be in fluid communication with the vacuum port and the fluid source by at least two different paths.

The manifold channel assembly further includes a plurality of connection channels, each of which extends from each of the plurality of branch channels to a plurality of wells, each of the plurality of connection channels being in fluid communication with the plurality of wells. I can.

Another aspect of the present invention,

Preparing a card layer having a plurality of wells disposed therein;

Disposing the card layer between the first film layer and at least one second film layer;

Bonding the first film layer to the first surface of the card layer; And

Including; bonding the second film layer to the second surface of the card layer,

The card layer, and at least one of the first film layer and the second film layer, forming the following (i) and (ii)

A method of manufacturing an array assembly can be provided:

(i) a vacuum channel extending between the plurality of wells and vacuum ports and/or in fluid communication with the plurality of wells and vacuum ports; And

(ii) an array fill channel extending between a plurality of wells and a fluid source, and/or in fluid communication with the plurality of wells and a fluid source.

The concave conduit of the card layer may have a manifold structure.

Another aspect of the present invention

(a) preparing a sample container including a reaction region in fluid communication with the array assembly;

(b) forming a reaction mixture by performing an analysis method on the sample in the reaction zone;

(c) opening a vacuum port to discharge air from a plurality of wells and a plurality of channels, and introducing a vacuum onto the array assembly;

(d) forming an evacuated array by sealing the vacuum ports so that the plurality of wells maintain a vacuum; And

(e) opening the connection opening to introduce the reaction mixture into a plurality of wells through an array filling channel; including,

The array assembly,

A plurality of wells configured in an array;

A connection opening between the reaction region and the array;

Vacuum port; And

A plurality of channels for allowing each well in the array to be in fluid communication with the connection opening and the vacuum port; including,

It can provide a method of using an array assembly for a sample.

Mixing the reaction mixture with one or more reagents disposed in each of a plurality of wells to form a second reaction mixture; And performing a second analysis method on the second reaction mixture.

The second reaction mixture may be a PCR mixture.

It may further include a step of thermally cycling the second reaction mixture.

It may further include; detecting an amplification product in at least one of the plurality of wells.

The sample may contain microorganisms, and one or more reagents may contain antibiotics.

The antibiotic is included at a first concentration in a first well of a plurality of wells, a second concentration is included in a second well of a plurality of wells, and the second concentration is lower than the first concentration. It can be a concentration.

Step (c) may provide a vacuum of 2 to 150 mba to the plurality of wells.

Steps (c) and (d) may be performed before step (b) is completed.

Another aspect of the present invention

(a) preparing a sealed container comprising a first reaction zone and a second reaction zone comprising a dried component;

(b) performing a first reaction in the first reaction zone to form a reaction mixture;

(c) performing the step (b) before or during the step of hydrating the dried component of the second reaction zone with a hydration fluid to form a hydrated component;

(d) adding a portion of the reaction mixture to the hydrated component; And

(e) performing a second reaction in a second reaction zone; containing,

It is possible to provide a method of performing a multistage biological reaction in a sealed sample container.

The second reaction region is an array of wells, each well contains a dried component, and at least some of the wells are different from the wells of other parts of the plurality of wells. Can have ingredients.

Each well of the well array may include a barrier layer that delays the entry of the hydration solution into the well.

The step of hydration may include filling a plurality of wells with a hydration solution, and then removing excess hydration solution from the well array.

A method of performing a multistage biological reaction in the sealed sample container comprises: transferring the reaction mixture to the array; A portion of the reaction mixture entering each well through a barrier layer; And removing the excess reaction mixture from the array.

A method of carrying out a multistage biological reaction in a sealed sample container comprises: transferring the mixture to the array; And sealing a part of the reaction mixture adjacent to each well outside the barrier layer so that the reaction mixture and the hydrated component are mixed through the barrier layer.

The barrier layer is a pierced layer having at least one opening per well, and the opening may have a size that makes it difficult for a fluid to pass through the opening without applying a slight force.

The first reaction may be a first step PCR, and the second reaction may be a second PCR.

The second reaction region may maintain a cool temperature before step (d).

Before performing the step (c), evacuating the at least partially evacuated second reaction region; may further include.

Another aspect of the present invention,

A card having a plurality of wells arranged in an array;

A connection opening on the first side of the card;

A channel system in fluid communication with the connection opening and a plurality of wells; And

Including; a vacuum port in fluid communication with the channel system,

Array assemblies can be provided.

The array includes a plurality of rows, each of the plurality of rows includes one or more wells, the channel system includes a manifold channel assembly,

The manifold channel assembly,

A plurality of branch channels extending along the plurality of rows, each of which is in fluid communication with a plurality of wells; And a first main channel in fluid communication with a connection opening extending along a first end of the plurality of rows,

The plurality of branch channels extend from the first main channel,

A second main channel in fluid communication with the plurality of branch channels,

The second main channel extends along the second end of the plurality of rows, the vacuum port communicates with the second main channel,

Each of the wells may be in fluid communication with the vacuum port and the connection opening by at least two different paths.

The array assembly

Further comprising a first film layer and a second film layer,

The first and second film layers may form a pouch, and the card layer may be at least partially sealed inside the pouch.

Another aspect of the present invention,

A plurality of reaction chambers connected to the fluid, and an array,

The array,

A connection opening in fluid communication with at least one of a plurality of reaction chambers connected to the fluid;

A plurality of reaction wells;

Vacuum port; And

In fluid communication with the connection opening, the plurality of wells, and the vacuum port, each of the reaction wells of the array is fluidly connected to the vacuum port by at least two paths, and each of the reaction wells of the array is at least 2 Including; a channel system connected to the fluid source by four paths,

A reaction vessel can be provided.

The vacuum port may be reversibly sealed, and the connection opening may be reversibly sealed.

Another aspect of the present invention,

A plurality of wells arranged in an array;

A vacuum channel in fluid communication with the plurality of wells and vacuum ports; And

Including; an array filling channel in fluid communication with the plurality of wells and the fluid source

Array assemblies can be provided.

The array assembly

A card layer having a plurality of wells formed therein;

A first film layer coupled to the first surface of the card layer and sealing the first end of each of the plurality of wells; And

Including; a second film layer bonded to the second surface of the card layer,

The vacuum port may include an opening in at least one of the first film layer and the second film layer.

At least one of the vacuum channel and the array filling channel may be formed in at least one of (i) the card layer, (ii) the second film layer, and (iii) the card layer and the third film layer. .

At least a portion of at least one of the vacuum channel and the array filling channel may be formed between the first film layer and the second film layer.

A part of the array filling channel may be formed between the first film layer and the second film layer, and may be disposed in at least a part of a perimeter of the card layer.

Further comprising a concave conduit in the card layer,

The concave conduit of the card layer is of a manifold structure, each well in the array is in fluid communication with the vacuum port by at least two paths, and each well will also be connected to the fluid source by at least two paths. I can.

The second film layer has a concave conduit formed therein,

Further comprising a third layer disposed between the second film layer and the card layer,

The third film layer may include a plurality of piercing portions extending therethrough, and the plurality of piercing portions may be in fluid communication with the concave conduit of the second film layer.

At least a portion of the array filling channel may be disposed between the concave conduit of the second film layer and the bond between the second film layer and the third film layer between two or more portions of the concave conduit of the second film layer. .

At least a portion of the vacuum channel may be provided as a part of the array filling channel, and may have a structure for maintaining the array filling channel in an open position.

An open seal may be further included in the array fill channel between the plurality of wells and the fluid source.

It may further include one or more reagents disposed in each of the plurality of wells.

The plurality of wells are disposed in each of the plurality of rows, and the array fill channel includes a fluid source and a connection channel in fluid communication with a manifold channel assembly,

The manifold channel assembly,

A plurality of branch channels extending along each of the plurality of rows, each of which is in fluid communication with the plurality of wells; And

Including; a first main channel extending along the first end of the plurality of rows,

The plurality of branch channels may extend from the first main channel, and the array filling channel may extend from the first main channel.

The manifold channel assembly further includes a second main channel in fluid communication with the plurality of branch channels,

The second main channel extends along the second end of the plurality of rows,

The vacuum port is in fluid communication with the second main channel,

Each of the wells may be in fluid communication with the vacuum port and the fluid source through at least two different paths.

The manifold channel assembly further includes a plurality of connection channels each extending from each of the plurality of branch channels to a plurality of wells,

Each of the plurality of connection channels may be in fluid communication with the plurality of wells.

Another aspect of the present invention

A sample introduction region, at least one first reaction region in fluid communication with the sample introduction region, a second reaction region in fluid communication with the first reaction region, the second reaction region including a plurality of wells, , The vacuum channel is in fluid communication with the plurality of wells and vacuum ports, the array fill channel extends between the plurality of wells and the first reaction region, and/or the array fill channel is in fluid communication with the plurality of wells and the first reaction region, Preparing a reaction vessel, wherein an open seal is disposed between the first reaction region and the second reaction region;

Performing at least one step of an analysis method on the reaction vessel;

Forming a vacuum partially on the plurality of wells to place at least a portion of the plurality of wells, the vacuum channel, and the array fill channel at a pressure lower than atmospheric pressure;

Sealing a portion of the vacuum channel and forming an exhaust array such that a vacuum is maintained in at least a portion of the plurality of wells and the array fill channel; And

Including, supplying the fluid to the exhaust array by opening an open seal disposed between the first reaction region and the second reaction region so that the fluid flows into the plurality of wells through the array filling channel.

It is possible to provide a method for forming an in-situ vacuum in a reaction vessel while performing the analytical method.

At least one step of the analysis method

Taking out a reaction vessel packaged at atmospheric pressure from an ambient pressure package, introducing a sample into the sample introduction region, performing the analysis method on the reaction vessel and partially introducing a vacuum to form an exhaust array Inserting into an apparatus configured for, performing one or more reactions in the first reaction zone, and preparing to supply fluid to the exhaust array.

The step of performing one or more reactions in the first reaction zone,

Decomposing a sample to form a decomposition product, separating the decomposition particles from the decomposition product, mixing the decomposition product and silica magnetic beads, collecting the nucleic acid by the silica magnetic beads, and moving the residual decomposition product to a waste chamber , Washing the magnetic beads at least once, moving the washing solution to a waste chamber, eluting nucleic acids from the silica magnetic beads, performing a single or complex first PCR reaction, and the second reaction zone In preparation for performing a second PCR reaction in a plurality of wells of, it may include any one of the steps of diluting the product of the first PCR reaction.

In the method for forming an in-situ vacuum in a reaction vessel while performing the analysis method, the reaction vessel comprises at least one reagent in fluid communication between at least one of the sample introduction region, the first reaction region, and the second reaction region. It may further include a blister.

The one or more reagent blisters contain dry reagents,

The vacuum forming method may further include forming a partial vacuum on the at least one reagent blister containing the drying reagent.

The method for forming an in-situ vacuum in a reaction vessel while performing the analysis method may further include mixing a fluid with the drying reagent to form a first mixture.

One or more of the reagent blisters may include reagents for sample preparation, nucleic acid recovery, first step PCR and second step PCR.

Another aspect of the present invention,

Including a reaction vessel and apparatus,

The reaction vessel includes a sample introduction region, at least one first reaction region in fluid communication with the sample introduction region, and a second reaction region in fluid communication with the first reaction region,

The second reaction region extends between the plurality of wells, the plurality of wells and the vacuum port, and/or the vacuum channel in fluid communication with the plurality of wells and the vacuum port, the plurality of wells and the first reaction region, and / Or an array filling channel in fluid communication with the plurality of wells and the first reaction region, and an open seal disposed between the first reaction region and the second reaction region,

The apparatus is configured to perform an analytical method using the reaction vessel, and comprises a vacuum system for introducing a partial vacuum to one or more portions of the reaction vessel during performing one or more steps of the analytical method.

System can be provided.

One or more portions of the reaction vessel configured to form a partial vacuum may be dried such that the partial vacuum is not formed against the partial pressure of water.

The partial vacuum of one or more portions of the reaction vessel configured to form a partial vacuum may be between 2 and 150 mba.

At least one portion of the reaction vessel configured to form the partial vacuum may include at least a portion of a plurality of wells, vacuum channels, and array fill channels.

The apparatus may include a sealing member that applies a seal to preserve a partial vacuum formed in one or more portions of the reaction vessel.

The reaction vessel may further include one or more reagent blisters in fluid communication with at least one of the sample introduction region, the first reaction region, and the second reaction region.

At least one of the one or more reagent blisters comprises a dry reagent,

One or more portions of the reaction vessel configured to form a partial vacuum may contain one or more reagent blisters containing the dry reagent.

The apparatus may be a PCR apparatus comprising at least one heater disposed and arranged to thermally cycle at least a portion of the reaction vessel.

The apparatus may include at least one heater disposed and arranged to control the temperature of at least a portion of the reaction vessel to perform an isothermal reaction.

The mechanism includes at least one heater disposed and arranged to control the temperature of at least a portion of the reaction vessel, one or more actuators for moving fluid to the reaction vessel, and a fluid in one or more portions of the reaction vessel. It may include one or more seals to control movement.

The above summary may be provided to introduce in a simplified form a selection of concepts that are further described in the detailed description below. The above summary is not intended to identify key features or essential features of the claimed subject matter, nor is it used to assist in determining the scope of the claimed subject matter. Additional features and advantages will be described in the following description, some of which are apparent from the description or can be seen by embodiments of the present invention.

Features and advantages can be realized and obtained by means of the claims particularly set forth in the appended claims and combinations thereof. These or other features may become more fully apparent from the following description and appended claims, or may be known by the embodiments of the present invention as described below.

An embodiment of the present invention may include an array assembly capable of being exhausted at the point of use and/or in real time (ie, in performing an analysis method). Thus, the vacuum is a few seconds to moisture (i.e. long enough to create a vacuum and reseal the system, preferably sufficient to open the fill seal to fill the evacuated chamber (e.g. a well array) with liquid. Can be maintained for a long time).

1 is a diagram showing a flexible pouch useful for self-contained PCR,
FIG. 2 is an exploded perspective view of a device including the pouch of FIG. 1 and for use with the pouch of FIG. 1,
FIG. 3 is a partial cross-sectional view of the FIG. 3A device including the bladder component of FIG. 2 with the pouch of FIG. 1;
4 is a diagram showing a motor used in an embodiment of the device of FIG. 2,
Figure 5a is a diagram showing another embodiment of a flexible pouch,
5B is a diagram showing a cross-sectional view of a portion of the pouch 5a taken along the line BB,
5C is a diagram showing a cross-sectional view of a portion of the pouch of FIG. 5A taken along the CC line,
6A is a diagram showing an embodiment of a two-stage array,
6B is a cross-sectional view taken along line BB of FIG. 6A illustrating an embodiment of a well filling system,
6C is another embodiment showing another embodiment of the well filling system,
7A is a diagram showing a two-stage array representing an embodiment of the array exhaust system at the point of use,
7B is a partial cross-sectional view of FIG. 7A taken along line BB,
8A is a perspective view showing an embodiment of an array assembly including a card,
Figure 8b is a detailed view of a part of the array assembly of Figure 8a,
8C is a plan view of another embodiment of an array assembly including a card layer in a pouch,
8D is a plan view of the pouch,
9A is an exploded perspective view of an embodiment of a two-stage high-density array with vacuum channels,
9B is a bottom view of the two-stage high-density array of FIG. 9A showing the configuration of the two-stage high-density array,
9C is a partial cross-sectional view of FIG. 9A taken along line CC.
10A to 10E are diagrams illustrating a method of initiating a reaction by rehydrating contents in a two-stage array well according to an embodiment,
Figure 10f is a diagram showing another method of initiating a reaction in the method shown in Figures 10a-10e,
11A is a schematic illustration of an apparatus configured to create a vacuum in a portion of a reaction vessel during or after performing one or more steps of an analytical method,
FIG. 11B is a diagram showing a part of the vacuum system and array of the reaction vessel of FIG. 11A configured to form a vacuum in-situ,
Figure 11c is a diagram showing the vacuum system and reagent blister of the reaction vessel of Figure 11a configured to form a vacuum in real time (in-situ),
12A to 12D are diagrams illustrating various array wells and channel types.

This application claims priority to Patent No. 62/510,682 filed on May 24, 2017, and the entire priority is incorporated by reference in the present invention.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings.

Since various forms and embodiments are possible without departing from the spirit and technology of the present invention, the present invention should not be construed as being limited to the exemplary embodiments described below. The exemplary embodiments described below are provided so that the present invention is thorough and complete, and may be provided to convey the scope of the present invention to those skilled in the art. In addition, the sizes and relative sizes of layers and regions in the drawings may be exaggerated for clarity. The same reference numerals refer to the same components throughout the specification.

Unless otherwise defined, all terms (including technical or scientific terms used herein) have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. In addition, terms such as terms defined in a commonly used dictionary should be construed as having a meaning consistent with their meaning in the context of this application and related technologies, and ideally or excessively unless explicitly defined in the present specification. It should not be interpreted. In the present specification, terms used in the description of the present invention are intended to describe only specific embodiments and are not intended to limit the present invention. A number of methods and materials similar or equivalent to those described herein may be used in embodiments of the present invention, and only certain exemplary materials and methods are described herein.

All publications, patent applications, patents, or other references mentioned herein are incorporated by reference in their entirety. In case of conflicting terms, the present specification will control.

Various aspects of the invention, including devices, systems, methods, and the like, may be described with reference to one or more exemplary embodiments. The terms “exemplary” and “for example” as used herein mean “to serve as an example, instance, or illustration,” and should not be construed as necessarily preferred or advantageous over other implementations disclosed herein. In addition, reference to the present disclosure or the "implementation" or "embodiment" of the present invention is included as a specific reference to one or more embodiments of the present invention, and vice versa, and the present invention It is intended to be provided as illustrative examples without limiting the scope of. This is indicated by the appended claims rather than by the following description.

It will be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a tile” includes one, two or more tiles.

Similarly, reference to multiple referents should be construed as including a single referent and/or a plurality of referents unless the content and/or context clearly dictates otherwise. Thus, reference to "tiles" does not necessarily require a plurality of such tiles. Instead, it will be understood that it has nothing to do with conjugation; More than one tile is considered here.

As used throughout this application, the words “can” and “may” refer to an acceptable meaning (ie, a possibility) rather than an essential meaning (ie, a meaning that must mean). Meaning). Also, "including", "having", "containing," characterized by", variations thereof (eg, "includes"," has ), includes, “contains”, etc.), and similar terms used herein, including the claims, are inclusive and/or open, “comprising” and variations thereof ( For example, comprise and comprises) may have the same meaning as the words, and do not exclude additional configuration or method steps that are not exemplarily mentioned.

As used herein, "top", "bottom", "left", "right", "top", "bottom", "top than", "bottom", "inner", "outer"," Directions and/or any terms such as "inner", "outer", "inner", "outer", "front", back end", "forward", "reverse", etc. are only intended to indicate a relative direction and/or direction May be used, and may not limit the scope of the invention, including the specification, invention and/or claims.

When a component is referred to as "coupled," "connected," or "connected" or "on" to another component, the component can be understood as a direct bond, connection, connection, or Constituent elements or constituent elements may exist between them. On the other hand, when a component is referred to as "directly connected", "directly connected" or "directly connected" or "directly on" another component, another component is present in the middle. It should be understood as not doing.

An exemplary embodiment of the concept of the present invention is described herein with reference to a cross-sectional view that is a schematic diagram of an ideal embodiment (and intermediate structure) of the exemplary embodiment. Thus, for example, it can be transformed from the urban structure as a result of manufacturing techniques and/or tolerances. Accordingly, the exemplary embodiments of the present invention should not be construed as being limited to the specific structure of the region shown in the present specification, and for example, in the case of a manufacturing method, it should be construed as including a structural deviation as a result. Accordingly, the areas shown in the drawings are schematic in nature and their shapes do not exemplify the actual shape for the area of the instrument and do not limit the scope of the exemplary embodiments.

Terms such as “first” and “second” may be used herein to describe various configurations, but these configurations are not limited by the above terms. The term is only used to distinguish one element from another. Therefore, the "first" configuration may be referred to as a "second" configuration that does not depart from the bar indicated by the present embodiment.

In addition, the embodiments described herein may be used in combination with other embodiments described or disclosed within the scope of the present invention. Accordingly, products, members, components, devices, devices, systems, methods, processes, compositions, and/or combinations thereof according to embodiments of the present invention are disclosed in this specification so as not to depart from the scope of the present invention (system, The features, features, components, members, components, steps and/or others described in another embodiment (including methods, devices, etc.) may be included, incorporated, or otherwise included. Some embodiments, or an aspect of the invention, may be described as an alternative. However, these alternatives may not be mutually exclusive. Accordingly, expressions such as “alternative” and “alternatively” may be replaced with “additional”, “additional”, and the like. Therefore, what is mentioned as a specific feature related to an embodiment of the present invention may not be limited to the above embodiment.

The headings used in this specification are for constitutive purposes only and are not meant to limit the scope of the description or claims. For ease of understanding, reference numerals have been used where possible to designate similar components in common in the drawings. Also, where possible, similar numbers have been used for components in the various drawings. In addition, each of the alternative structures of a particular component may include a separate character added to the component number.

In the present specification, the term “about” is used in the meaning of about, almost, or a region around it. When the term “about” is used in connection with a numerical range, it can be viewed as modifying the range by extending the perimeter above and below the numerical value.

In general, in the present specification, the term "about" is used to correct a numerical value above and below the stated value by an error of 5%. When this range is expressed, other embodiments may include one specific value and/or another specific value. Similarly, when a numerical value is expressed as an approximation using the above "about", it will be understood that the specific value forms another embodiment. Further, it will be appreciated that the thresholds in each range are all important with respect to the other thresholds.

The word “or” used herein may include one of a specific group and a combination of the group.

By “sample” meaning an animal; The tissue or organ; Cells (also cells inside the subject, taken directly from the subject, or cultured or cultured from cells); Cell lysate (or lysate fraction) or cell extract; Solutions comprising one or more molecules derived from longitudinal, cellular material or material or viral material (eg, polypeptide or nucleic acid); Or it may mean a solution containing a non-naturally occurring nucleic acid analyzed as described herein. In addition, the sample may be a host or pathogenic cell, cell component, or bodily fluid or secretion that may or may not contain nucleic acids (e.g., blood, urine, feces, saliva, tears, bile, or sedimentary fluid, but is not limited thereto. Not). Samples may also include, but are not limited to, environmental samples such as soil, water (fresh water, wastewater, etc.), air monitoring system samples (e.g., material trapped in air filter media), surface cotton wool, and media. have.

As used herein, “nucleic acids” refers to naturally occurring or synthesized oligonucleotides or polynucleotides, regardless of whether DNA or DNA-RNA hybrids, single or double helix, sense or antisense. Referred to as a nucleotide (polynucleotide), which can be hybridized to a complementary nucleic acid by a Watson-Crick base pair.

In addition, the nucleic acids of the present invention are nucleotide analogues (eg, BrdU) and non-phosphate diester nucleoside bonds (eg, peptide nucleic acid (PNA) or thiodiester bonds) It may include. In particular, nucleic acids may include, but are not limited to, DNA, RNA, mRNA, rRNA, cDNA, gDNA, ssDNA, dsDNA, and combinations thereof.

“Probe”, “primer” or “oligo nucleotide” may refer to a single helical nucleic acid molecule of a defined sequence capable of base pairing with a second nucleic acid molecule containing a complementary sequence (“target”). The stability of the resulting hybrid can vary depending on the length, GC content and the degree of base pairing that occurs. The degree of base pairing can be influenced by parameters such as the degree of complementarity between the probe and the target molecule and the degree of severity of the hybridization conditions. The degree of hybridization severity can be influenced by parameters such as temperature, salt concentration, and concentration of organic molecules such as formamide, and can be determined by methods known to those skilled in the art. Probes, primers, and oligonucleotides may be labeled radioactively, fluorescently or non-radioactively detectably by methods well known to those skilled in the art. dsDNA binding dyes can be used to detect dsDNA. A “primer” is specifically configured to be extended by a polymerase, while a “probe” or “oligo nucleotide” may or may not be configured to be extended by a polymerase.

The "dsDNA binding dye" may refer to a dye that generally fluoresces more strongly and thus exhibits fluorescence more differentially when bound to double-stranded DNA than when it becomes single-stranded DNA or when there is no solution. While reference is made to dsDNA binding dyes, any suitable dye may be used, with some non-limiting examples to the dyes described in US Pat. No. 7,387,887, which can be used in the present invention, which is incorporated herein by reference. Other single product materials can be used to detect nucleic acid amplification and melting, practical enzymes, antibodies and the like known in the art.

“Specific hybridization” means that a probe, primer or oligonucleotide recognizes and physically interacts (ie, base pairing) with a substantially complementary nucleic acid (eg, a sample nucleic acid) under very harsh conditions, and substantially base pairing with another nucleic acid. It could mean not achieving

“Very harsh conditions” can typically mean that the about melting point (Tm) occurs at -5° C. (ie, 5° below the Tm of the probe). Functionally, very harsh conditions can be used to identify nucleic acid sequences with at least 80% sequence identity.

While PCR is an amplification method used in an embodiment of the present invention, any amplification method used for primers may be suitable. Such a combined method, polymerase chain reaction (PCR); Strand displacement amplification (SDA); Nucleic acid sequence-based amplification (NASBA); Cascade rolling circle amplification (CRCA); Loop-mediated isothermal amplification of DNA (LAMP); Isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN); Target-based-helicase dependent amplification (HDA); Transcription-mediated amplification (TMA); Etc.

Thus, when the term PCR is used, it should be understood to include other alternative methods of amplification. For amplification methods that do not include individual cycles, the reaction time can be formed by cycle, doubling time, or crossing point (Cp), and additional reaction time will be added by adding an additional PCR cycle in the examples described herein. I can. The protocol can be adjusted accordingly.

“Cross point (Cp)” (or alternatively, cycle critical point (Ct), quantitative cycle point (Cq), or synonyms used in the art) as used herein is an experimentally determined, given PCR product ( For example, it may mean the number of cycles of PCR required to obtain a fluorescence signal above some threshold for a target or an internal standard). The cycle in which each reaction rises above the critical point depends on the amount of target (ie, reaction template) present when the PCR reaction is initiated. The threshold value may typically be set at a point where the fluorescence signal of the product is detectable than the background fluorescence signal; However, other threshold values may be applied. As an alternative to setting a somewhat arbitrary threshold, Cp can be determined by calculating the response point at which the first, second or nth order derivative has a maximum value, which can determine the cycle in which the curvature of the amplification curve has a maximum value. have. An exemplary derived method is disclosed in US Pat. No. 6,303,305, the entirety of which is incorporated by reference. On the other hand, as long as the same threshold is used for all reactions being compared, the location or method of setting the threshold may not be important. Other grain boundary values known in the art may be used and may be substituted for Cp, Ct or Cq in any of the methods described herein.

Various examples of the invention refer to human targets and human pathogens, but the above examples are only illustrative. The methods, kits and devices described herein can be used to detect and analyze a wide range of nucleic acid sequences from a wide range of samples, including human, veterinary, industrial and environmental.

Various embodiments disclosed herein use a self-contained nucleic acid assay pouch to analyze a sample for the presence of various biological materials, such as antigens and nucleic acid sequences, for example in a single closed system. US Pat. No. 8,394,608, which is incorporated herein by reference, of such a system comprising a pouch and a device for use with the pouch; And US Patent Application No. 2014-0283945 disclosed in more detail. However, such a pouch is only an example, and the nucleic acid preparation and amplification method described in the present invention is a 96-well plate, various open or closed system samples known in the art including plates, arrays, carousels, etc. Can be carried out in a container.

“Sample well”, “amplification well”, “amplification container, etc.” are used in the present invention, and the terms refer to wells, tubes and various other reaction vessels used in such amplification systems. You can already do what you include.

In one embodiment, the pouch can be used to analyze multiple pathogens. The pouch may contain one or more blisters, for example, used as sample wells in a closed system. For example, various steps are optionally disposable, including nucleic acid preparation, primary bulk complex PCR, dilution of primary amplification products, and post-amplification analysis such as secondary PCR, ultimate selective real-time detection or degradation curve analysis. It can be performed in a pouch. In addition, it may be understood that various steps may be performed within the pouch of the present invention, but one or more steps may be omitted for a specific application, and the structure of the pouch may be changed accordingly.

As used herein, "point-of-use" refers to a step in which an apparatus or method is performed by the system immediately before or during use by the system.

For example, creating a vacuum in one or more assay device chambers at the “point of use” means that one or more assays can be performed either immediately prior to performing the assay using the assay device (eg, within an hour) or using the assay device. Means that a vacuum is formed during or after real time. This is in contrast to carrying out steps such as establishing a suitable vacuum in the manufacture of the assay device and then storing the device under the appropriate vacuum until immediately before use.

1 illustrates an exemplary pouch 510 that may be used in various embodiments or may be reconfigured for various embodiments. The pouch 510 is similar to US Patent No. 8,895,295 of FIG. 15, and the same reference numerals are used for the same configuration. The fitment 590 is provided with entry channels 515a to 515l serving as a reagent reservoir or waste reservoir.

Illustratively, reagents may be lyophilized in fitment 590 and rehydrated prior to use. Each channel 514, 538, 543, 552, 553, 562, and 565 would be similar to the same number of blisters 522, 544, 546, 548, 564, and 566 of U.S. Patent No. 8,895,295 of FIG. I can. The second stage reaction region 580 of FIG. 1 is similar to the reaction region of U.S. Patent Application No. 8,895,295, but the second stage wells 582 of the high density array 581 are arranged in a somewhat different pattern.

The more circular pattern of the high-density array 581 of FIG. 1 can remove the well of the coater, and consequently fill the second-stage well 582 more uniformly. As shown, the high density array 581 is provided with 102 second stage wells 582. Pouch 510 may be suitable for use in a FilmArray® (BioFire Diagnostics, LLC, Salt Lake City, UT) instrument. However, the pouch embodiment may be merely exemplary.

Other containers may be used, but by way of example, the pouch 510 is a flexible plastic film or polyester, polyethylene terephthalate (PET), polycarbonate, polypropylene, polymethyl methacrylate and mixtures thereof, and combinations thereof. It can be formed of two layers of the same other analogous material, and can be formed of layers that can be produced by any process known in the art, including extrusion, plasma deposition and lamination. For example, each layer may be composed of one or more material layers of a single structure or more than one structure stacked together.

In addition, metal foils or plastics with aluminum lamination can be used. Other barrier materials that can be sealed together to form blisters and channels are known in the art. If a plastic film is used, the layers can be bonded to each other, for example by heat sealing. For example, the material may have low nucleic acid binding capacity.

In embodiments used for fluorescence monitoring, plastic materials with sufficiently low absorbance and autofluorescence at the operating wavelength may be desirable. These materials can be identified by testing plastic differences, plasticizer differences, composite ratios, and thickness differences of the film. In the case of plastics with aluminum or other foil lamination, the portion of the pouch to be read by the fluorescence detection device can be left without the foil. For example, when fluorescence is monitored in the second stage well 582 of the second stage reaction region 580 of the pouch 510, one layer or two layers of the well 582 will be left without the foil. I can.

For example, for PCR, a film composed of polyester (Mylar, DuPont, Wilmington DE) about 0.0048 inches (0.1219 mm) thick and polypropylene 0.001 to 0.003 inches (0.025 to 0.076 mm) thick. Lamination can be activated. For example, the pouch 510 may be made of a transparent material capable of transmitting about 80% to 90% of incident light. In an exemplary embodiment, the material may be transferred between the blisters by applying pressure to the blisters and channels, for example air pressure. Thus, in an embodiment where pressure is applied, the exemplary pouch material may be flexible enough to have a desired effect by pressure.

The term “flexible” in the present invention may be used to describe the physical characteristics of the pouch material. The term "flexible" in the present invention is defined as being easily deformed without cracking, breaking, crazing, etc. at the pressure level used in the present invention.

For example, thin plastic sheets such as Saran™wrap and Ziploc®bags and thin metal foils such as aluminum foil are flexible. However, even in embodiments using pneumatic pressure, flexibility may only be required for specific areas of the blister and channel. Also, flexibility may be required on only one side of the blister and channel, as long as the blister and channel can be easily deformed. Another area of the pouch 510 may be made of a rigid material, or may be reinforced with a rigid material. Therefore, when a “flexible pouch” or “flexible sample container” is used, only a portion of the pouch or sample container may require flexibility.

For example, a plastic film may be used for the pouch 510. The metal sheet may be milled or otherwise cut with aluminum or other suitable material, for example, to form a die having a pattern on the surface. Illustratively, when mounted in a pneumatic press controlled to a working temperature of 195º (e.g. A-5302-PDS, Janesville Tool Inc., Milton WI), the pneumatic press works like a printing press so that the die contacts the film. In some cases, it can melt the sealing surface of the plastic film. As such, the plastic film used as the pouch 510 may be cut and welded together using a laser cutting machine and a welding device. Like PRC primers (eg placed on a film and dried), antigen binding substrates, magnetic beads, and zirconium silicate beads can be sealed inside various blisters by forming a pouch 510. Reagents for sample processing may be placed entirely or separately on the film prior to sealing. In one embodiment, nucleotide triphosphates (NTPs) are located on the film separately from the polymerase and primer, so that the activity of the polymerase can be essentially removed until the reaction is hydrated by the aqueous sample. have. If the aqueous sample is heated before being hydrated, this creates conditions for hot-start PCR, and can reduce or eliminate expensive chemical hot-start components. In another embodiment, the constituents may be provided in a powder or pill structure, and may be placed in a blister prior to final sealing.

The pouch 510 can be used in a similar manner to that described in US Pat. No. 8,895,295. As an example, 300 μl of a mixture containing a sample to be tested (100 μl) and a digestion buffer (200 μl) may be injected into an injection port (not shown) of the fitment 590 adjacent to the entry channel 515a, The sample mixture may be introduced into the entry channel 515a. In addition, water may be injected into a second injection port (not shown) adjacent to the entry channel 515l of the fitment 590, and distributed through a channel (not shown) provided in the fitment 590, each It is possible to hydrate 11 different reagents previously provided in a dried structure to the entry channel 515b through the entry channel 515l. An exemplary method and apparatus for injecting a sample and injecting a hydration solution (e.g., water or buffer) is disclosed in U.S. Patent Application 2014-0283945, the entirety of which is incorporated herein by reference. do. However, the method and apparatus are only an example, and other methods of introducing the sample and hydration liquid into the pouch 510 may be within the scope of the present invention.

Illustratively, the reagent may include a freeze-dried PCR reagent, a DNA extraction reagent, a washing solution, an immunoassay reagent, and other chemical components. Exemplarily, the reagent may be a reagent for nucleic acid extraction, a first step complex PCR, a complex reaction dilution, a control reaction, and a second step PCR preparation. In the embodiment shown in FIG. 1, everything required for injection may be a sample in one injection port and water in another injection port. After injection, the two injection ports can be sealed. For detailed information on the pouch 510 and the fitment 590 having various structures, refer to US Patent No. 8,895,295, which is incorporated by reference.

After injection, the sample may be transferred from the injection channel 515a to the disintegration blister 522 through the channel 514. Decomposition blister 522 is provided as beads or particles 534, such as ceramic beads or other abrasive elements. In addition, the sample is configured to vortex at high speed through impact using a rotating blade or paddle of the FilmArray® mechanism. By shaking, vortexing, sonicating, and similar sample processing in the presence of decomposed particles such as zirconium silicate (ZC) beads 534, bead-milling can be an effective method to form decomposition products. have. As used herein, terms such as “lyse”, “lysing” and “lysate” are not limited to ruptured cells and include destruction of non-cellular particles, such as viruses. can do.

FIG. 4 shows a beating motor 819, comprising a blade 821 that can be mounted on a first side 811 of a support member 802 of the instrument 800 shown in FIG. 2. The blade may extend through the slot 804 to contact the pouch 510. However, the motor 819 may be mounted on other structures of the device 800. As an exemplary embodiment, the motor 819 may be a Mabuchi RC-280SA-2865 DC motor (Chiba, Japan) mounted on the support member 802. In one exemplary embodiment, the motor may rotate at 5,000 to 25,000 rpm, and as a more detailed example, it may rotate at 10,000 to 20,000 rpm, and as a more detailed example, it may rotate at about 15,000 to 18,000 rpm. have. For the Mabuchi motor, it has been found that 7.2V provides enough rpm for disassembly. However, when the blade 821 applies an impact to the pouch 510, the actual speed may be slightly slower. Other voltages and speeds depending on the motor and paddle used can be used for disassembly. Optionally, a controlled small amount of air may be provided into the blader 822 adjacent to the decomposition blister 522. In some embodiments, it has been found that during the disintegration process, when placing and supporting the disintegration blister, one or more small amounts of air aid partially fill the adjacent bladder. Alternatively, other structures, for example a rigid or flexible gasket around the disintegration blister 522, or other holding structure, may be used to hold the pouch 510 during the disintegration process. Further, the motor 819 is only an example, and can be used to mill, swing or rotate the sample at high speed. In some embodiments, chemicals or heat may be used in addition to or instead of mechanical degradation.

When the sample material is properly degraded, the sample can be transferred to the nucleic acid extraction region. For example, it may be moved to blister 546 through channel 538, blister 544 and channel 543. The transferred sample may be mixed with a nucleic acid-binding substrate such as silica 533 coated with magnetic beads. Alternatively, the magnetic beads may be rehydrated, for example, using a fluid provided from one of the entry channels 515c to 515e, which is then moved through the channel 543 to the blister 544, and thereafter. , It may be moved to the blister 522 through the channel 538. The mixture can be allowed to incubate for an appropriate period of time, for example about 10 seconds to 10 minutes. A shrinkable magnet located within a device adjacent to the blister 546 traps the magnetic beads 533 from the solution, thereby forming a pellet against the inner surface of the blister 546. If incubation occurs inside blister 522, several portions of the solution may be required to be moved to blister 546 for capture. The solution may then be moved from the blister 546 and moved back through the blister 544 into the blister 522 used as a waste container. One or more wash buffers may be provided to the blister 546 from one or more injection channels 515c-515e through blister 544 and channels 543. Optionally, the magnet is retracted and the magnetic bead 533 can be cleaned by moving the bead back and forth from the blisters 544 and 546 through the channel 543. When the magnetic beads 533 are washed, the magnetic beads 533 are recaptured in the blister 546 by activation of the magnet, and the cleaning liquid may be transferred to the blister 522. The process may be repeated as necessary to wash the digestion buffer and the sample residue from the nucleic acid-binding magnetic beads 533.

After washing, the elution buffer stored in the injection channel 515f is moved to the blister 548, and the magnet can be contracted. The solution is circulated between the blisters 546 and 548 through the channel 552 to crush the pellets of the magnetic beads 533 in the blister 546, and the captured nucleic acid is decomposed from the beads and mixed in the solution. The magnet is activated once again to capture the magnetic beads 533 in the blister 546, and the eluted nucleic acid solution is transferred to the blister 548.

The first step PCR master mixing from the injection channel 515g may be mixed with the nucleic acid sample in the blister 548. Optionally, the mixture may be mixed by forcing the mixture between blisters 548 and 564 through the channel 533. After mixing several cycles, the solution may be stored in a blister 564, the first step PCR primer pellet is provided as at least one primer set for each target, and the first step complex PCR is performed. Can be. If there is an RNA target, the RT step may be performed before or simultaneously with the first step complex PCR. Exemplarily, in the FilmArray® apparatus, the first step complex PCR temperature cycle may be 15 to 20 cycles. However, depending on the specific application needs, other levels of amplification may be desirable. The first step PCR master mix can be any of a variety of master mixes known in the art. In an exemplary embodiment, the second step PCR master mixing may be any one of the chemicals disclosed in U.S. Patent No. 9,932,634 incorporated by reference for use with a PCR protocol of 20 seconds or less per cycle.

After the first step PCR is performed with the required number of cycles, the sample may be diluted. Illustratively, the sample may be forcibly put back into the blister 548 and diluted by adding a second step PCR master mix from the injection channel 515i, leaving only a small amount in the blister 564. Alternatively, the diluted buffer solution may be transferred from the injection channel 515i to the blister 566, after which the fluid is moved back and forth between the blisters 564 and 566 to be amplified in the blister 564. It can be mixed with the sample. If desired, the dilution can be repeated several times, using the dilution buffer from infusion channels 515j and 515k. Or, by way of example, the injection channel 515k may be reserved for PCR analysis after the sequence or other, after which the second step PCR master mixing is added as part or all of the diluted amplified sample from the injection channel 515h. Can be. The dilution level can be determined by varying the number of dilution steps, or by varying the number of dilution steps, or components for amplification, although other components may be appropriate, especially for non-PCR amplification, for example, a dilution buffer or agent comprising polymerase, dNTPs and appropriate buffer The two-step PCR master mix can be adjusted by changing the percentage of discarded sample prior to mixing. If desired, the mixture of the sample and the second step PCR master mix may be preheated in a blister 564 before being transferred to the second step well 582 for two step amplification. This preheating may eliminate the need for hot-start components (antibodies, chemicals or anything else) in the second stage PCR mixture.

The exemplary second stage PCR master mix is incomplete and lacks primer pairs, and each of the 102 second stage wells 582 may be preloaded with a specific PCR pair. If desired, the second stage PCR master mix is free of other reaction components, and these components can be preloaded into the second stage well 582. Each primer pair may be similar or the same as the first step PCR primer pair, or may overlap within the first step primer pair. Transfer of the sample from blister 564 to second stage well 582 completes the PCR reaction. Once the high density array 581 is filled, each second stage reaction may be sealed to each second stage blister by any number of means, as is known in the art. An exemplary method of filling and sealing high density array 581 without cross contamination is disclosed in US Pat. No. 8,895,295, incorporated by reference. Illustratively, the various reactions in the wells of the high-density array 581 are thermally cycled simultaneously or individually with one or more Peltier devices, illustratively, although other means for thermoelectric cycling are known in the art.

In a specific embodiment, the second step PCR master mix may include a dsDNA binding dye LCGreen®Plus (BioFire Diagnostics, LLC) to generate a signal representing amplification. However, the dye is only exemplary, and as known in the art, other signals including fluorescent, radioactive, chemiluminescent, enzyme-labeled dsDNA binding dyes and probes may be used. Alternatively, the wells 582 of the array 581 may be provided without a signal, and the results may be reported through a post-processing process.

When air pressure is used to move the material into the pouch 510, in one embodiment, a “bleeder” may be used. A part of the bladder assembly 810 is illustrated in FIGS. 2 and 3. The bladder assembly 810 includes a bladder substrate 824 containing a plurality of expandable bladders 822, 844, 846, 848, 864, and 866, each of which is individually expandable and , Exemplarily, expandable by a compressed gas source. Since the bladder assembly 810 can accommodate the compressed gas and can be used multiple times, the bladder assembly 810 may be made of a material stronger or thicker than the pouch. Alternatively, bladders 822, 844, 846, 848, 864, and 866 may be formed from a series of substrates fixed together with gaskets, sealing members, valves and pistons. Alternatively, arrays or mechanical actuators and closure members can be used to seal the channels and direct fluid movement between the blisters.

To be suitable for the apparatus of the present invention, mechanical sealing systems and actuators are described in detail in the full text of WO 2018/022971, which is incorporated herein by reference in its entirety in WO 2018/022971.

The success of the second PCR reaction may depend on the template generated by the complex first step reaction. Typically PCR can be performed using high purity DNA. Methods such as phenol extraction or commercial DNA extraction kits can provide high purity DNA. Samples processed through the pouch 510 may require adjustment to compensate for lower purity. PCR can be inhibited by a biological sample component, and the biological sample component can be a potentially impaired person. Illustratively, hot-start PCR, higher concentration of Taq polymerase enzyme, MgCl ₂ concentration adjustment, primer concentration adjustment, and addition of adjuvants (DMSO, TMSO, or glycerol) are selective to compensate for lower nucleic acid purity. Can be used as The purity issue may be of greater concern in the first stage amplification, but can be similarly adjusted for the second stage amplification.

When the pouch 510 is positioned inside the device 800, the bladder assembly 810 is pressed against one surface of the pouch 510 to inflate a specific bladder, the pressure increases the liquid to the pouch 510 ) Will pressurize to come out of the blister. In addition to the bladder corresponding to the plurality of blisters of the pouch 510, the bladder assembly 810 may have an additional pneumatic actuator such as a bladder or a pneumatic driven piston corresponding to the various channels of the pouch 510. I can.

2 and 3 are exemplary multiple pistons or hard seals 838, 843, 852, 853, and 865 corresponding to the channels 538, 543, 553, and 565 of the pouch 510, and the fitment ( 590) Shows seals 871, 872, 873, 874 that minimize backflow into the interior. When activated, the hard seals 838, 843, 852, 853, and 865 form a pinch valve to pinch off and close the channel.

To form liquid within a specific blister of pouch 510, the hard seal can be activated across the channel leading to the blister and the channel leading from the blister, and the actuator can function as a pinch valve to close the channel. have. Illustratively, in order to mix two volumes of liquid in different blisters, a pinch valve actuator that pushes the connecting channel is activated, and the pneumatic bleeder on the blister is alternately pressurized to connect the blister to the channel. The liquid is mixed by moving the liquid back and forth.

The pinch valve actuator may have various structures and sizes, and may be configured to pinch off one or more channels at a time. Although pneumatic actuators are described herein, linear sputter motors, motor driven cams, rigid pedals driven by pneumatic, hydraulic or electromagnetic forces, rollers, rocker-arms and, in some cases, cocked springs. Other methods of providing pressure to the pouch including various electromechanical actuators such as springs) can be used.

In addition, there are various methods of reversibly or irreversibly closing the channel other than applying a vertical pressure to the vertical axis of the channel. These include twisting bags across channels, heat sealing, rolling actuators, and various valves sealed within channels such as butterfly valves and ball valves. In addition, a small Peltier device and other temperature control mechanism can be placed in a position adjacent to the channel and set to a temperature sufficient to freeze the fluid, effectively forming a seal.

In addition, the structure of FIG. 1 is suitable for an automated mechanism characterized by an actuator element positioned in each of the blister and channel, but the actuator can be held in a stationary state, and the pouch 510 is used for sample analysis and nucleic acid preparation. It can be converted into a small number of actuators that can be used in several treatment regions, including treatment regions for other applications of the pouch 510, such as, first and second step PCR and immuno-assay and immuno-PCR.

The rollers that apply to the channels and blisters can be of a particularly useful structure for the pouch 510 to convert between different treatment areas. Therefore, while the pneumatic actuator is used in the embodiment of the present invention, when the "pneumatic actuator" is used in the present invention, it can be understood that other methods of providing different actuators and pressures depending on the structure of the pouch and mechanism may be used. have.

Referring to FIG. 2, each pneumatic actuator may be connected to a compressed air source 895 through a valve 899. Although several hoses 878 are shown in FIG. 2, it can be understood that each pneumatic fitting is connected to the compressed gas source 895 through one hose 878. The compressed gas source 895 may be a compressor, or alternatively, a compressed gas cylinder such as a carbon dioxide cylinder.

Compressed gas cylinders can be particularly useful when portability is required. In addition, other sources of compressed gas may be used within the scope of the present invention. As shown in the embodiments of FIGS. 12 to 16, similar air pressure control may be provided to regulate the fluid in the pouch 1400, or other actuators, servos, etc. may be provided.

Several other components of the instrument 810 may be connected to the compressed gas source 895. Illustratively, the magnet 850 mounted on the second surface of the support member 802 is deployed and contracted using gas that has come out of the compressed gas source through the hose 878. Other methods of moving the magnet 850 are known in the art. The magnet 850 is located in a recess 851 of the support member 802. The recess 851 may be a passage through the support member 802 so that the magnet 850 may contact the blister 546 of the pouch 510.

However, when the magnet 859 is deployed, the magnet 850 is close enough to provide a sufficient magnetic field to the blister 546, and when the magnet 850 is fully retracted, the magnet 850 is If it does not significantly affect any magnetic beads 533 in the blister 546, depending on the material of the support member 802, the concave portion 851 needs to extend completely through the support member 802. There is no. Although described above as shrinking the magnet 850, an electromagnet may be used, and the electromagnet may be activated by controlling the flow of electricity through the electromagnet. Thus, while this specification describes the extension or contraction of a magnet, this term may be wide enough to cover other methods of extending the magnetic field.

The pneumatic connection member may be a pneumatic hose or a pneumatic air manifold, thereby reducing the number of hoses or valves required. Similar magnets and methods for activating magnets may be used in the embodiments of FIGS. 12-16. Thus, while this specification describes withdrawal or withdrawal of a magnet, it is understood that this term is broad enough to encompass other methods of withdrawing a magnetic field. It is understood that the pneumatic connection may be a pneumatic hose or a pneumatic air manifold, thereby reducing the number of hoses or valves required. It is understood that similar magnets and methods for activating magnets may be used in the embodiments of Figures 1 and 2.

In addition, the various pneumatic pistons 868 of the pneumatic piston array 869 are connected to a compressed gas source through a hose 878. Although only two hoses 878 are shown connecting the pneumatic piston 868 to the compressed gas source 895, each pneumatic piston 868 may be connected to the compressed gas source 895. Twelve pneumatic pistons 868 are shown.

A pair of temperature control elements can be mounted on the second side 814 of the support member 802. As used herein, "temperature control element" refers to a device that applies heat to or removes heat from a sample. The temperature control element according to an embodiment of the present invention may include a heater, a cooler, a Peltier device, a resistance heater, an induction heater, an electromagnetic heater, a thin film heater, a printing element heater, a positive temperature coefficient heater, and a combination thereof. It is not limited. The temperature control element may include a number of heaters, coolers, Peltier, and the like.

In one aspect, a given temperature control element may include one or more heaters or coolers. For example, the temperature control element according to an embodiment of the present invention may include a Peltier device to which a separate resistance heater is applied to an upper surface and/or a lower surface of the Peltier. Although the term “heater” is used herein, other temperature control elements may be used to control the temperature of the sample.

As previously described, the first stage heater 886 may be positioned to heat or cool the contents of the blister 564 for the first stage PCR. As shown in Fig. 2, the second stage heater 888 is positioned for heating or cooling the contents of the second stage blister 582 of the array 581 of the pouch 510 for the second stage PCR. Can be. However, the heaters can also be used for other heating purposes, and other heaters can be included to make them suitable for a particular application.

As described above, a Peltier device that is thermally cycled between two or more temperatures is effective for PCR, while in some embodiments it may be desirable to keep the heater at a constant temperature. Illustratively, this can be used to reduce the operating time by eliminating the time required to convert the heater temperature beyond the time required to convert the sample temperature.

In addition, this arrangement can improve the electrical efficiency of the system, as it only requires thermally cycling the smaller sample and sample container, not a larger (having more thermal mass) Peltier device. For example, the appliance may include a plurality of heaters (e.g., two or more) positioned to be connected to the pouch to achieve thermal circulation, and set temperatures for heat treatment, elongation, and denaturation, for example. . Two heaters may be sufficient for many applications. In various embodiments, to achieve thermal circulation, the heater may be moved, the pouch may be moved, or a fluid may be moved relative to the heater. For example, the heater may be arranged in a linear or circular arrangement. With reference to the second step PCR, a suitable heater type has been described above.

When fluorescence detection is required, an optical array 890 may be provided. As shown in FIG. 2, the optical array 890 may include a light source 898, and illustratively may include a filtered LED light source, filtered white light or laser illumination, and a camera 896. have. The camera 896 may have a plurality of photodetectors corresponding to the second stage well 582 of the pouch 510 by way of example.

Alternatively, the camera 896 captures an image including the second-stage well 582, and the image may be divided into individual regions corresponding to each of the second-stage wells 582. Depending on the structure, the optical array 890 may be static, or the optical array may be placed on a vehicle attached to one or more motors and moved to obtain a signal from each of the individual second stage wells 582. .

On the other hand, other arrangements may be possible. The second stage heater according to the embodiment of the present invention shown in FIG. 18 provides a heater on the opposite side of the pouch 510 shown in FIG. 2. This orientation is merely exemplary, and the orientation can be determined by spatial constraints within the device. When the second reaction region 580 is made of an optically transparent material, the photodetector and the heater may be disposed on either side of the array 581.

As shown, the computer 894 can regulate the valve 899 of the compressed air source 895, and thus, all air pressures of the instrument 800. In addition, many pneumatic systems of the device may be replaced with mechanical actuators, pressure application means, etc. in other embodiments. Computer 894 can also control heaters 866 and 888 and optical array 890.

Each of these configurations may be electrically, illustratively connected by a cable 891, and physical or wireless connection may be possible within the scope of the present invention. Computer 894 may be housed within instrument 800 or may be external to instrument 800. Further, the computer 894 may include a built-in circuit board that controls some or all of the configurations, and may also include an external computer such as a desktop or notebook to receive and display data from the optical array. An interface, for example, a keyboard interface including a key for inputting information and variables such as temperature, circulation time, etc. may be provided. Also, as an example, a display 892 may be provided. The display 892 may be, for example, an LED, an LCD or other display.

Another conventional instrument represents PCR in a sealed flexible container. For example, reference can be made to US Pat. Nos. 6,645,758, 6,780,617, and 9,586,208, which are incorporated herein by reference by reference. However, including cellular degradation in a sealed PCR container can improve ease of use and safety, especially if the sample to be tested contains a biohazard. In an embodiment of the invention, waste from cell degradation and waste from all other steps remain in the sealed pouch. The pouch contents can be removed for further testing.

2 shows an exemplary instrument 800 that may be used with the pouch 510. The instrument 800 may include a support member 802 that forms a wall of the casing or is mounted within the casing. The instrument 800 may also include a second support member (not shown) that is selectively movable relative to the support member 802 to allow for retraction and removal of the pouch 510. For example, the lid may cover the pouch 510 when the pouch 510 is inserted into the device 800. In another embodiment, both of the two supporting members may be fixed while the pouch 510 is fixed in place by other mechanical means or pneumatic pressure.

In an exemplary embodiment, the heaters 886 and 888 may be mounted on the support member 802. However, this arrangement is only an example, and other arrangements may be possible. Exemplary heaters may include Peltiers, other block heaters, resistance heaters, electromagnetic heaters, and thin film heaters known in the art to thermally cycle the contents of the blister 864 and the second stage reaction region 580. Bladder plate 810 having bladders 822, 844, 846, 848, 864, 86, hard seals 838, 843, 852, 853 and seals 871, 872, 873, 874 The further assembly 808 may be formed, and the bladder assembly may be mounted on a movable support structure capable of moving toward the pouch 510, for example, as the pneumatic actuator is disposed in contact with the pouch 510. have.

The pouch 510 can be inserted into the instrument 800, and when the movable support member is moved toward the support member 802, the various bristers of the pouch 510 may be used for the various bladders of the bladder assembly 810 and Arranged to be adjacent to the various seals of the assembly 808, accordingly, activation of the pneumatic actuator pressurizes liquid from one or more blisters of the pouch 510 or forms a pinch valve in one or more channels of the pouch 510 do.

The relationship between the blister and channel of the pouch 510 and the relationship between the bladder and the seal of the assembly 808 are shown in more detail in FIG. 3.

5A shows another embodiment of a pouch 5000 (also referred to herein as a'science card'). The pouch 5000 may be used in various embodiments or may be reconstituted for various embodiments described for PCR, microbial experiments, or other various tests in the present invention.

The pouch 5000 may be configured to be used in a device disclosed in WO 2017/147085, which is incorporated by reference of the present invention, or may be configured to be used in a variety of other devices. The exemplary pouch 5000 of FIG. 5A may include a plurality of regions or blisters in which sample preparation, nucleic acid amplification, and detection may occur.

The exemplary pouch 5000 is a sample preparation blister 5005, a first PCR blister 5010, in which a sample containing a nucleic acid to be amplified and analyzed, can be introduced into the pouch, the first before the second step. A second stage PCR array 5081 comprising a scalpel dilution well 5015 for measuring a portion of the product from the stage PCR, and a plurality of individual reaction wells 5082.

The scalpel well 5015 may also be in fluid communication with the blisters 5020 and 5025, and a reagent for the second step PCR may be introduced into the contents of the dilution well 5015 and then mixed. As an example, a sample for the second step PCR may be prepared by repeatedly mixing the contents of the scalpel well 5015 with a reagent for the second step PCR between the blisters 5020 and 5025. The second stage array 5081 may also be in fluid communication with the waste container 5035. Alternatively, blister 5010 may be used for sample preparation and first step PCR, and blister 5005 may be used as a waste container, eg, a waste container for sample preparation waste.

The blisters 5005, 5010, 5020, and 5025, the dilution well 5015, and the second stage array 5081 may be fluidly connected by channels 5050a-5050e. Samples and reagents may enter the pouch 5000 through entry channels 5040a-5040f and entry ports 5045a-5045f. Alternatively, the pouch 5000 may be equipped with a device similar to the fitment 590 of FIG. 1 in order to introduce samples and reagents into the pouch 5000. In addition, the pouch 5000 may contain a reagent that is dehydrated (eg, lyophilized) in a fitment or similar structure, and the dehydrated reagent is used in an appropriate hydration buffer before using the pouch. Can be hydrated. In another embodiment, a liquid reagent may be drilled into the pouch 5000.

In one embodiment, the pouch 5000 is made of a material having a plurality of layers that are sealed together to form the pouch 5000 (a plurality of layers composed of the same material or a material composed of different types of material and having a plurality of layers). Can be manufactured from

In Figures 5B and 5C, cutaway views are shown along lines B-B and C-C showing layers of material, which can be used to fabricate other portions of pouch 5000. In the first region of the pouch 5000 illustrated in FIG. 5B, the illustrated pouch 5000 may be manufactured from the first layer 5090 of the film bonded to the second layer 5098 of the film. The first and second layers 5090 and 5098 may be bonded to each other in any conventional manner known in the art. For example, it may be joined by heating and pressing, ultrasonic welding or laser welding, but is not limited thereto.

5B also shows that the pouch 5000 leaves an open area between the first and second layers 5090 and 5098, and defines the perimeter of the open area with a sealed margin along the opening, It shows that the emitter or channel can be formed within the pouch. -An exemplary welding is shown in 5084 in FIGS. 5A and 5B.

5C shows another area of the pouch 5000 containing a thick thick card material that can be used to form the wells of the second stage array 5081. The region of the pouch 5000 shown may be manufactured from a first film layer 5090, a pressure-sensitive adhesive layer 5092, a card layer 5094, a second pressure-sensitive adhesive layer 5096, and a second film layer 5098. have.

As an exemplary embodiment, the wells 5082 of the second stage array 5081 may be formed in the card layer 5094. Channels (e.g., channels 5040c) and blisters (e.g., blisters (e.g., blisters) by leaving open spaces between the film layers (e.g., first layer 5090 and second layer 5098) 5005), as shown in FIG. 5B, the card layer 5094 can be extended, and the blister and/or channels make an appropriate cutout in the card layer 5094. It can be formed by forming.

Similarly, the channels 5050a-5050e and the entry channels 5040a-5040f may be formed by forming a cutout in either of the first or first pressure-sensitive adhesive layers 5092 and 5096, and other structures may be possible. . Although the exemplary blister area has flexibility, the card layer 5094 may optionally be a rigid material with less flexibility. However, it can still be understood as part of a flexible sample container.

Thus, a “flexible pouch” can be understood as requiring flexibility only in a specific area. Alternatively or additionally, the fluid channels between the blister regions add another film layer, piping, rigid layer on top of the first film layer 5090 or below the second film layer 5098, and weld the layers together. And can be formed by leaving open blister regions and channels between the layers.

Other materials may be used, but for example, the film layer of pouch 5000 may be formed from a flexible plastic film or other flexible material similar to pouch 510 shown in FIG. 1. For example, the pouch 5000 may be made from materials such as polyester, polyethylene terephthalate (PET), polycarbonate, polypropylene (PP), polymethyl methacrylate, combinations, mixtures, and laminated layers thereof. Can, but is not limited to this Further, the pouch 5000 may be manufactured by any method known in the art including extrusion, plasma deposition, and lamination.

A similar material (e.g., polycarbonate) may be used for the card layer 5094. Also, other materials including plastic or metal foil with aluminum lamination may be used. Other barrier materials that can be sealed together to form blisters and channels are known in the art. If a plastic film is used, the layers may be bonded to each other, for example, by heat sealing or laser welding. Illustratively, the material has low nucleic acid binding capacity. If fluorescence detection is used, an optional transparent material may be used in an appropriate area of the pouch (eg, near the second stage array).

In addition to or instead of the examples of film materials described above, a barrier film may be used in one or more layers to form the pouch 5000 or any of the other pouches described herein. For example, barrier films may be desirable in some embodiments because they have lower water vapor and/or oxygen permeability than low conventional plastic films.

For example, typical barrier films may have a water vapor transmission rate (WVTR) of about 0.01 g/m ² /24h to about 3 g/m ² /24h, preferably about 0.05 g/m ² /24h to about 2 g /m ² /24h (e.g., within about 1 g/m ² /24), and the oxygen permeability may be from about 0.01 cc/m ² /24h to about 2 cc/m ² /24h, preferably May be from about 0.05 cc/m ² /24h to about 2 cc/m ² /24h (for example, within about 1 cc/m ² /24h).

The blocking film according to the embodiment is a metal by vapor deposition of a metal (for example, aluminum or other metal) or by sputtering coated with an oxide (for example, Al ₂ O ₃ or SiO _x ) or other chemical components. It may include a film that can be converted, but is not limited thereto.

A typical example of a metallized film is aluminum-coated Mylar, and the aluminum-coated Mylar is a metal-coated biaxially oriented PET (BoPET). In some applications, the coated barrier film may be laminated with a layer of polyethylene, PP or similar thermoplastics, provide sealability, and improve fracture resistance. Like a conventional plastic film, the barrier film layers used to manufacture the pouch may be bonded to each other. Illustratively, it may be bonded by heat sealing. Illustratively, the material may have low nucleic acid binding and low protein binding ability. Other barrier materials are known in the art that can be sealed together to form blisters and channels.

The pouch 5000 may be used in a manner similar to that described above for the pouch 510, and/or may be used in a manner similar to that described in US Pat. No. 8,895,295.

Referring to FIG. 5A, two alternative sequences are described for filling the pouch and preparing a sample, performing a first step PCR, and performing a second PCR.

As a first exemplary method, sample preparation and first step PCR may be performed in separate blisters. The first exemplary method is referred to as “a method having three regions” in the present invention, wherein the three regions may be sample preparation, first step PCR, and second step PCR. In the following embodiments of the “three zone method” and “two-zone method”, the pouch 5000 is an embodiment of a pouch, and a different pouch structure May be suitable for a method with three regions and/or two regions.

In the first step, the sample may be injected into the blister 5005 through the filling channel 5040a. In one embodiment, cells, viruses, etc. may be degraded in the blister 5005 using the wiping system described in detail in another embodiment of the present invention.

Alternatively, cellular degradation can be achieved by alternative degradation devices or chemical degradation. In this case, the decomposition device may be an ultrasonic device or a bead beater, but is not limited thereto. Optionally, disassembly may be assisted by heating the sample (e.g., about 70-90°C) with one or more heater elements of the heater assemblies detailed in another embodiment of the present invention. After decomposition, the sample is cooled to about 0°C to about 20°C (e.g., about 10-15°C) by means of a thermoelectric cooling element (e.g., Peltier element), e.g. silica coated Nucleic acid recovery can be aided using the magnetic beads. Other cooling elements may include, but are not limited to, a fluid or gas heat exchange element, a fan-cooled heat sink, a heat transfer tube, and a condensing mechanism.

The magnetic beads may be injected into the blister 500 through the filling channel 5040a or 5040b for use in recovering nucleic acids from the degradation product. Alternatively, the digested cells, digestion beads, magnetic beads, digestion buffer, and the like may be injected simultaneously or sequentially into the blister 5005 before digestion.

Illustratively, the magnetic beads and the decomposition product may be mixed in a low temperature state (e.g., by adjusting the temperature of one of the heaters, for example, in a temperature range of about 0-10 ° C. When the magnetic beads and the decomposition product are thoroughly mixed for a sufficient time, the magnetic beads may be agglomerated in the blister 5005 by a magnet exemplarily provided in the apparatus, and the used decomposition product is liquid waste through the channel 5040b. Can be sent to

Thereafter, the washing buffer may be injected through the filling channel 5040a. The washing buffer and magnetic beads may be mixed at a low temperature (about 0-10°C). The magnetic beads may be agglomerated again, and the used washing buffer may flow into the liquid waste through the channel 5040b. The washing cycle may be repeated one or more times.

After washing, the elution buffer (optionally including the first step PCR primer) may be injected into the blister 5005 through the filling channel 5040a. The elution buffer (and the first step PCR primer) and the magnetic beads may be selectively mixed in a high temperature state (about 70-90 °C), and may be, for example, controlled by one or more heaters.

In the case of the first-stage PCR, PCR master mix (eg, polymerase, dNTPs, and other amplification components known in the art) may be injected into the blister 5010 through the filling channel 5040c. The PCR master mix can be heated (e.g., about 57 °C) before introducing the eluate from the magnetic beads. In the blister 5005, the magnetic beads may be agglomerated again, and the eluate may be sent to the blister 5010 through the channel 5050a.

In one embodiment, the first step PCR may be performed in a blister 5010 having a rotational motion of a wiper system disclosed in WO 2017/147085, which is incorporated by reference, under temperature control by two heaters, by way of example. .

Alternatively, the first stage PCR thermocycling is performed under the control of one heater of a heater assembly in which the blister 5010 comprises two different heaters at two different temperatures (e.g., heat treatment temperature and denaturation temperature). Then, it may be performed by converting the heater assembly or pouch 5000 to be under the control of another heater.

The channels flowing into and out of the blister 5010 may be closed while performing the first step PCR, exemplarily, may be closed with a seal similar to the seal shown in FIGS. 2 and 3. In some embodiments, the speed of the first PCR in the pouch may be increased by using a volume reduction protocol. For example, the volume reduction protocol is a step of performing PCR in several cycles (e.g., 1 to 10 times) in the initial volume (e.g., 100 μL) of the blister 5010, It may include reducing the volume by about half, performing PCR with more cycles (eg, 5-10), and reducing the beak of the blister 5010 by about half. . Since a smaller volume of liquid has a smaller thermal mass and has a faster thermal cycle than a larger volume of liquid, the volume reduction can reduce the cycle time for the PCR reaction.

After cycling the first-stage PCR a sufficient number of times (e.g., 20-30 cycles), a small sample (e.g., ~1-5 μL) of the first-stage PCR is diluted through a channel (channel 5050b). It can be sent to well 5015 and channels 5050c-5050e can be closed.

A sample for the second step PCR may be prepared by injecting the second step PCR master mix into the blister 5025 through the channel 5040e. The seal channels 5050b and 5050e can be closed, and the seal 5050c alc 5050d can be opened.

Further, to dilute the first step PCR product for second step PCR, the samples in well 5015 can be mixed with the master mix by mixing between blisters 5025 and 5020 and well 5015.

Blisters 5020 and 5025 and wells 5015 may be heated before or during mixing for a physical “hot-start”. The channel 5050e is then opened, and the seals 5050c and 5050d can be closed. Through this, the second-stage PRC mixing may be transferred to the second-stage PCR array 5081. In other embodiments, the pouch 5000 may include one or more additional dilution wells and sets, the set being mixed blister ?? It may be downstream from well 5015 and upstream from blisters 5025 and 5020 and array 5081. For example, in some embodiments involving a concentrated first step PCR primer or highly concentrated product, it may be desirable to dilute the first step primer and product larger enough to be achieved with one dilution well. Thermal cycling for the second stage PCR in the array 5081 can be achieved by exemplarily converting the heater assembly back and forth as detailed in the present invention.

In the second exemplary method, sample preparation and first step PCR can be performed in the same blister. This is referred to as a "two zone method" in the present invention, and the sample preparation and first step PCR may be performed in the first region, and the second step PCR may be performed in the second region. .

In the first step, the sample may be injected into the blister 5010 through the filling channel 5040c. As an example, cells, viruses, and the like may be degraded in the blister 5010 using the wiping system described in detail in another embodiment of the present invention. Alternatively, cellular degradation can be achieved by alternative degradation devices or chemical degradation. In this case, the decomposition device may be an ultrasonic device or a bead beater, but is not limited thereto.

Disassembly may be assisted by heating the sample by raising the temperature with one or more heater elements of the heater assembly described in detail in other embodiments of the present invention. After decomposition, the sample is optionally with a thermoelectric cooling element (e.g., Peltier element) to reduce the temperature (e.g., reduce it to an ambient temperature such as 0-10°C, but is not limited thereto). Can be cooled.

The magnetic beads may be injected into the blister through the filling channel 5040c to recover the nucleic acid from the decomposition product. As an example, after decomposition, the magnetic beads and decomposition products may be mixed at a low temperature (about, 0-10°C). In another embodiment, the combination of the degraded cells, the digestion buffer, the magnetic beads, and the selectively digestion beads may be injected together into a blister, through which the digestion and the nucleic acid encapsulation may occur almost simultaneously.

When the magnetic beads and the decomposition product are thoroughly mixed for a sufficient time, the magnetic beads may agglomerate in the blister 5010 together with the magnet, and the used decomposition product is passed through the channel 5050a through the blister 5005 (e.g., Liquid waste blister). Thereafter, the washing buffer solution may be injected into the blister 5010 through the filling channel 5040c. Optionally, the washing buffer and magnetic beads may be cooled at a low temperature (eg, about 0-10° C.).

The magnetic beads may be re-aggregated, and the used washing buffer may be introduced into the blister 5005. The washing cycle may be repeated more than once, and if desired, nucleic acids after washing may be eluted from the beads by injecting an elution buffer (and a first step PCR primer) into the blister 5010 (optionally in a heated state. At, for example about 70-90°C). The magnetic beads and (if present) any residual decomposition beads may be collected into the upstream half of blister 5010 and sent to waste blister 5005 through channel 5050a.

During the first-stage PCR, the wiper system may be set, and the first-stage PCR master mix may be injected into the channel 5040d, and if hot start is desired, the temperature is selectively raised (about 57°C). Can be maintained in The first step PCR master mixing may be mixed with a primer and a template, and the first step PCR may be performed as described above.

After the first step PCR, the protocol may proceed to the second step PCR, as described above for the method having three regions.

If fluorescence detection is required, an optical array can be provided. The optical array may include a light source, eg, a filtered LED light source, a filtered white light or laser illumination, and a camera 896. The camera may have a plurality of photodetectors corresponding to the second stage well 5081 of the pouch 5000 by way of example.

Alternatively, the camera may take an image including a second-stage well, and the image may be divided into individual regions corresponding to each of the second-stage wells. Depending on the structure, the optical array may be static, or the optical array may be placed on a vehicle attached to one or more motors and moved to obtain signals from each of the individual second stage wells. On the other hand, other arrangements may be possible.

6A, an array 6000 of wells that may be used for the second step PCR is shown in more detail. Array 6000 may be an independent array, or may be included as a group of wells in a larger array, such as part of array 5081.

Array 6000 may include individual wells 6002, 6004, 6006, 6008, and 6010. Each of the individual wells 6002, 6004, 6006, 6008, and 6010 can be used for a second step PCR reaction. In the illustrated embodiment, the wells 6002, 6004, 6006, 6008, and 6010 may be fluidly connected to the filling channel 6012; Holes 6014, 6016, 6018, 6020, and 6022 are formed in the fill channel 6012 to fill each well.

In one embodiment, the second stage array (e.g., 6002, 6004, 6006, 6008, and 6010 wells) may be partially in a vacuum state to facilitate the introduction of fluid from the filling channel 6012 into the well. . In one embodiment, the wells 6002, 6004, 6006, 6008, and 6010 may be sealed from the fill channel 6012 and each other, the leakage of fluid between the wells, which may be referred to as "cross action" Or mixing can be minimized or prevented by applying a seal (eg, heat seal) or pressure to or around the area shown at 6024.

Thus, a single seal may be applied to the region indicated by 6024 to close the wells 6002, 6004, 6006, 6008, and 6010 from the fill channel 6012 and between the wells to prevent cross-action. The cross-sectional structure of the array 6000 and a fluid passage for filling the wells are shown in Fig. 6b alc 6c. Array 6000 is shown as five wells 6002, 6004, 6006, 6008, and 6010 with respect to fill channel 6012, although more or less reaction wells may be associated with fill channels, and multiple The filling channels of can be connected with multiple well populations. Multiple arrays 6000 may be joined to form a larger array.

6B and 6C, FIG. 6B is a cross-sectional view taken along line B-B of FIG. 6A, and FIG. 6C is an alternative embodiment showing another embodiment of a well filling system. A portion of the array 6000 shown in cross section in FIGS. 6B and 6C may be made of a layer similar to that shown in FIG. 5C; It should be noted that the array 6000 may be included as part of the pouch 5000 shown in FIG. 5A in place of the array 5081. The well 6006b shown in FIG. 6B includes a first film layer 6030b, a second film layer 6032b, an adhesive layer 6034b, a card layer 6036b in which a well 6006b can be defined, and a second adhesive layer ( 6038b) and a third film layer 6040b. The well 6006c shown in FIG. 6C is very similar to the first film layer 6030c, the second film layer 6032c, the adhesive layer 6034c, the card layer 6036c in which the wells 6006c can be defined, It may be defined as the second adhesive layer 6038c and the third film layer 6040c. An important difference between the 6b and 6c is the difference in how wells 6006b and 6006c are formed around the layer and how wells are filled. I can.

In FIG. 6B, the fill channel 6012b may be formed by leaving a gap between the first and second film layers 6030b and 6032b through which liquid may flow. 6C shows a similar fill channel 6012c formed by leaving a gap between the first and second film layers 6030c and 6032c. The fill channel can be defined by a welding line 6026b or 6026c sealing the first and second film layers together around the array. An embodiment of how such welding is applied is shown in 6a.

As shown in FIG. 6A, the well welded portion 6026 includes an outer welded portion 6026a and an inner welded portion 6026b defining a filling channel 6012, and may include a space around the well. In FIG. 6B, the filling hole 6018b may be formed by forming a selective cutout in the second film layer 6032b and the first adhesive layer 6034b. In FIG. 6C, the filling hole 6018c may be formed by forming a selective cutout of the second film layer 6032c adjacent to the corresponding cutout of the first adhesive layer 6034c.

In Figure 6b, the well fill channels flowing around and filling the wells 6006b to fill the wells are formed by forming cutouts 6042b in the card layer 6036b and cutouts 6044b in the second adhesive layer 6038b. Can be formed. On the other hand, other methods of forming these channels may be possible. The design of the well fill channel of FIG. 6B can help to suppress cross-action between wells, for example because the fluid passages are complex. Likewise, since the fill channel 6012b and the fill hole 6018b are formed between the two film layers 6030b and 6032b, access to the fill hole 6018b and the well 6006b is, for example, a heat seal device or pressure By, for example, it can be sealed by a bladder. The bladder may expand over an area adjacent to 6024 in FIG. 6A or over all or part of the array 6000.

In Figure 6c, the well filling channel (6018c) flows directly into the well (6006c), the filling hole (6018c) is formed by forming a cutout (6046c) in the first adhesive layer (6034c) in fluid communication with the well (6006c) Can be. The fill design of FIG. 6C is also expected to generally inhibit crossover between wells. However, it can be sealed with a heat sealing device, for example, which can provide a better sealing than pressure alone.

In some embodiments, the wells of the second stage array may be at least partially under vacuum to facilitate filling the wells with fluid for the second stage PCR. In general, this can mean that the pouch is entrained in partial vacuum from the point of manufacture until the packaging around the sample container is opened at the point of use.

In one exemplary embodiment, Figures 7A and 7B illustrate an embodiment of a second stage array that can be used as an alternative to vacuum storage while still allowing reliable well filling in the second stage array. As will be described in detail below, FIGS. 7A and 7B illustrate an array including a vacuum method in which a partial vacuum can be formed in a liquid filling channel allowing a vacuum to be formed in an in-situ array. Schematically shows.

7A and 7B are another embodiment of a second stage array 11000 that can be filled without having a pouch manufactured or stored under partial vacuum. The second stage array 11000 is similar to the second stage array 6000 shown in FIG. 6A. The second stage array 11000 defined in part by the welding line 11026 may be provided with a unique second stage primer pair, and components for the second stage PCR (e.g., first stage PCR, It may include a second stage well (11002, 11004, 11006, 11008 and 11010) that can be filled with a diluted product produced from polymerase, dNTPs, etc., and described in detail in another embodiment of the present invention. Can be thermally cycled for two-step analysis.

The second stage well is filled from a fill channel 11012 in fluid communication with the fluidic vias 11052a to 1052e and fill holes 11014, 11016, 11028, 11020 and 11022 associated with each second stage well. Can be.

It is an exemplary vacuum passage 11050 that is integrally formed in the fill channel and in fluid communication with each fill hole, via, and second stage well. The vacuum passage 11050 may be in fluid communication with a port 11051 that may be disposed in a portion of the pouch spaced from the array. In an embodiment, the force 11051 may include a hole 11051a for providing a fluid connected to the vacuum passage. For example, the hole 1105a may be formed in either the layer 11030 or the layer 11032.

The vacuum passage 11050 may be used to create a partial vacuum on the second stage well in-situ (eg, by a vacuum pump in the instrument while the pouch is operating). Thus, the pouches need not be manufactured or stored under vacuum. In one embodiment, the vacuum channel 11050 may be connected to a remote vacuum hub on a pouch that may be connected to a vacuum source by way of example.

Referring to FIG. 7B, a cross section of the filling channel 11012 and the vacuum passage 11050 is shown. In the illustrated embodiment, the filling channel 11012 may be formed as an open space between the two film layers 11030 and 11032 bonded to each other at the edge of 11026 (eg, by laser welding). . In the illustrated embodiment, the vacuum passage 11050 may be formed as a sub-channel of one of the two film layers 11030 or 11032. In the illustrated embodiment, the vacuum passage 11050 is designed to keep the channel 11012 open and connect the fill holes, vias, and second stage wells to the vacuum source through the vacuum passage 11050 and the port 11051. It can have an arcuate shape.

In the absence of the vacuum channel 11050, there may be a tendency for the channel 11012 to collapse or “kiss” when a vacuum builds up in the channel to prevent evacuation of the well. In one embodiment, the vacuum channel 11050 may be formed as a concave conduit in one of the layers having a heat-forming fixture (eg, a hot'debossing' wire of a suitable shape).

In other embodiments, the vacuum channel 11050 may be formed by laser etching, gyrography, or the like. As shown in FIG. 7B, as an example of the heat forming channel, the vacuum passage 11050 has an arcuate shape that supports plastic and maintains the channel in an open state so that the vacuum can introduce air from the second-stage well.

Although the arch shape is shown in the illustrated embodiment, channels having other shapes used to keep the filling channel open may be used in the present invention. However, the vacuum passage 11050 may be sealed. For example, the vacuum passage 11050 may be sealed by bonding the layers 11030 and 11032 to each other by applying a heat seal on the vacuum passage 11050.

In one embodiment, a vacuum of at least 1 to 150 mbar (e.g., 2-10 mabr or more preferably 2 to 5 mbar) is applied to a second stage array (or vacuum portion formed in-situ). May be formed for 10 to 120 seconds on one or more portions of the reaction vessel (e.g., forming a vacuum in one or more portions of the reaction vessel and carrying out the reaction in the reaction vessel, or performing an analytical method). In an apparatus configured to conduct the reaction immediately prior to inserting the reaction vessel into the apparatus for use

After forming a vacuum, the vacuum channel can be sealed (eg, by heat), and the vacuum can be released at port 11051. Thus, the wells of the array may be under partial vacuum. Experiments on a prototype array having a vacuum channel similar to the vacuum channel described above show that forming a vacuum in situ is effective only for manufacturing or storing a pouch in a vacuum state.

Some embodiments in accordance with the present invention include a plurality of wells arranged in an array, a channel system in fluid communication with the plurality of wells and integrally formed (e.g., formed in a mold), and a channel system with the channel system. It communicates and may include a vacuum port integrally formed (eg, formed in a mold).

The channel system may also include a fluid opening separate from the vacuum port and integrally formed. In some embodiments, a fluid reservoir or source having an open seal (eg, a flexible seal, a peelable seal, or other seal known in the art) may be positioned adjacent the array assembly.

In some embodiments, a vacuum may be applied to the array assembly by creating a vacuum at the vacuum port; The open seal between the fluid source and the array assembly can typically remain closed while evacuating the array assembly. By opening the seal separating the fluid source from the array assembly, the fluid sample can be introduced into the plurality of wells through a vacuum passing through the channel system.

8A is an exemplary array assembly 8000 according to an embodiment of the present invention, which may be used as part of a pouch or card described in another embodiment of the present invention. Or, it shows an exemplary array assembly 8000 according to an embodiment of the present invention that can be used as part of any pouch or card discussed in other such embodiments.

As shown in FIG. 8A, the array assembly 8000 may include a card or card layer 8002 having a plurality of wells 8004 arranged in an array 8001.

As an example, for example, in the drawings of FIGS. 8C and 8D, the card 8002 may be placed in a reaction vessel sealed between two or more film layers. The card 8002 according to the embodiment includes a first (lower) surface 8016, a second (upper) surface 8018 facing the first surface 8016, and a peripheral outer portion 8020 extending therebetween. Includes.

Card 8002 can have any suitable size including thickness, length, width, and the like. For example, the size of the card 8002 may have a thickness of about 0.2 to 0.3, and the well may be set such that the card thickness and the well diameter are 0.20 to 0.5 μl, for example, 0.3 μl. . As shown in FIG. 8A, the card 8002 according to the embodiment may have a rectangular shape having a selectively rounded corner.

However, in other embodiments, the card 8002 may have any suitable shape. The wells 8004 may be arranged in several rows as shown in FIG. 8A, may be arranged in a circular or hexagonal shape as in FIGS. 1 and 2, or may be arranged in other configurations. In the present invention, the term "array" may include such an arrangement of wells.

Well 8004 may include, form, or be defined a hole or hole extending through card 8002 (eg, from first surface 8016 to second surface 8018 ). In an alternative embodiment, the hole or opening may only partially extend through the card 8002.

Accordingly, in some embodiments, the well 8004 may include, be formed, or may be formed by a recess or an indentation of the card 8002 or the second surface 8018 of the card. The recess or indentation forming the well 8004 is any within (or less) the thickness of the card 8002 (or between the first surface 8016 and the second surface 8018). Can have an appropriate depth of.

As shown in FIG. 8A, the well 8004 may have a circular or cylindrical structure. However, in alternative embodiments, the well 8004 may have other shapes or structures, some of which are shown in FIGS. 12A-12D.

As noted above, the array 8001 may have any suitable structure. As in the embodiment illustrated in FIG. 8A, the array 8001 may include a plurality of wells 8004 arranged in a plurality of columns 8022. For example, wells 8004a, 8004b, and 8004c are in first row 8082a of well 8004. The second row 8022b of well 8004 is adjacent to the first row 8022a of well 8004. The third row 8022c of the well 8004 is adjacent to a side opposite to the side adjacent to the first row of the second row 8022b. Additional rows 8022 of well 8004 are adjacent to, aligned (parallel), and/or the first to first row 8022a, second row 8022b and/or third row 8022c, and/or the first to It can be placed between the third row or on the opposite side. Thus, exemplary rows 8202 of well 8004 may be arranged in a grid or grid-like structure (e.g., each row 8022 of well 8004 is another row 8022 of well 80040). Can be arranged in or almost aligned).

As shown in FIG. 8A, the array 8001 of wells 8004 may include 10 rows 8202 of 10 wells 8004. However, in alternative embodiments, the array 8001 may have any suitable row 8022, e.g., more than 100 rows, 1 to 100 rows, 2 to 80 rows, 3 to 75 rows, It may have 4 to 60 rows, 5 to 50 rows, 6 to 40 rows, 7 to 20 rows, 8 to 12 rows, or any suitable number of rows or a range of rows between the above ranges. In some embodiments, the array 8001 is at least 1 row, 2 rows, 3 rows, 4 rows, 5 rows, 6 rows, 8 rows, 10 rows, 12 rows, 16 rows. , Can have 24 rows, 32 rows, 48 rows, 64 rows, or 96 rows.

In some embodiments, the row or plurality of rows 8022 may have any suitable number of wells 8004. For example, more than 100 wells, 1 to 100 wells, 2 to 80 wells, 3 to 75 wells, 4 to 60 wells, 5 to 50 wells, 6 to 40 wells, 7 to 20 wells, 8 to 12 wells , Or any suitable number of wells or a range of wells in between. In some embodiments, the row or plurality of rows 8022 is at least 1 well, 2 wells, 3 wells, 4 wells, 5 wells, 6 wells, 8 wells, 10 wells, 12 wells. , 16 wells, 24 wells, 32 wells, 48 wells, 64 wells or 96 wells. In at least one embodiment, the array 8001 may have at least one row 8042 of a plurality of wells 8004, and a plurality of rows 8042 or a plurality of wells of at least one well 8004 8004) of the plurality of rows 8082.

The array assembly 8000 includes a plurality of wells 8002, a fluid entry opening 8010 on the first side 8012 of the card 8002, and a vacuum port 8008 on the second side 8014 of the card 8002. It may further comprise a channel system 8006 in fluid communication with.

Thus, as described above, the fluid opening 8010 can be separated from the vacuum port 8008. In the illustrated embodiment, the vacuum port 8008 may be disposed on the opposite surface of the well 8004 from the fluid opening 8010. However, the orientation is only an example, and other structures may be possible. 8C-8D, the array assembly 8000 may be contained within a pouch or science card, and the fluid opening 8010 may be in fluid communication with other reaction zones.

As shown in FIG. 8A, the vacuum port 8008 may include a hole 8008a disposed in the card 8002. The hole 8008a may extend from the first surface 8016 to the second surface 8018 through the card 8002. Similar to the well 8004, in an alternative embodiment, the hole 8008a may only partially extend through the card 8002.

Accordingly, in some embodiments, the hole 8008a may include the card 8002 or a concave portion or a recess portion of the second surface of the card, or may be formed or defined by the concave portion or the recess portion. The recess or recess forming the hole 8008a may have any suitable depth within (or less) the thickness of the card 8002 (or between the first surface 8016 and the second surface 8018). have. As shown in FIG. 8A, the hole 8008a may have a circular or cylindrical structure. However, the hole 8008a may alternatively have a different shape or structure. In alternative embodiments, vacuum port 8008 may include an end or other portion of channel system 8006.

8A, the channel system 8006 can include a manifold channel assembly 8024 in fluid communication with a fluid entry channel 8034. The manifold channel assembly 8024 includes a first main channel 8026 in fluid communication with the fluid entry channel 8034, a second main channel 8028 in fluid communication with the vacuum port 8008, and a first main channel 8026. ) And the second main channel 8028 may include a plurality of branch channels 8030 extending between them. Each branch channel 8030 may extend parallel to one or more rows 8022 of well 8004.

In an embodiment, the first branch channel 8030a extends along the first column 8022a, the second branch channel 8030b extends along the second column 8022b, and the third branch channel 8030c is It may be extended in a manner that extends along the third row 8022c. A plurality of connection channels 8032 extend from each branch channel 8030 to each well 8004 in each row 8022.

For example, each connection channel 8032 may extend from one well 8004 of a specific column 8022 to a branch channel 8030 extending along the specific column 8022. Illustratively, each well 8004 of a particular row 8022 may be in fluid communication with a branch channel 8030 extending along a particular row 8222 by a respective connection channel 8032.

As shown in Fig. 8B, the connection channel 8032 and the branch channel 8030a (and virtually all channel systems 8006) have a recess or recess of the card 8002 or the second surface 8018 of the card. May include, and may be formed or defined by a recess or an inlet.

Channel system 8006 or the recesses or indentations forming one or more components of the channel system within (or less than) the thickness of the card 8002 (or between the first surface 8016 and the second surface 8018). ) Can have any suitable depth.

As shown in FIG. 8A, the channel system 8006 may have a rectangular structure. However, in alternative embodiments, the channel system 8006 may have other shapes or structures. In some embodiments, certain components of the channel system 8006 may have a greater or lesser depth than other components of the channel system 8006. However, as shown in FIG. 8A, all components of the channel system 8006 may have the same depth, and the well 8004 may have a greater depth.

12A-12D, examples of various well fill paths for wells in an array are shown. 8A to 8C illustrate an example of a "convoluted" array well fill path including a well 8004, an array fluid channel 8030, and a straight well fill channel 8032 as a well fill path. .

12A to 12D illustrate examples of a well filling path that is more indirect than the well filling channel 8202. Each of FIGS. 12A-12D includes wells 1200a-1200d and array fluid channels 1230a-1230d.

12A shows an embodiment of a helical channel 1232a leading to the well 1204a. 12B shows a zigzag channel 1232b leading to well 1204b. 12C shows a channel 1232c comprising a helical portion 1208c and a zigzag portion 1210c leading to a well 1204c. 12D shows a channel 1232d having a helical and zigzag portion 1208d and a parallel zigzag portion 1210d leading to a well 1204d. When placed in a pouch, the wells and channels shown in FIGS. 8A-8C and 12A-12D will be sealed with at least one layer on top and bottom.

Thus, the fluid path to the well may only be through a fluid channel and a well channel. Some pathways are more "convoluted" than others, but the combination of fluid channels and well channels generally separates each of the wells and minimizes mixing (i.e., cross-action) between wells as wells are filled. . The above-described example is only an example of a convoluted well filling path, and other well filling paths may be used.

Referring again to FIG. 8A, the channel system 8006 according to the embodiment may also include a fluid connection channel 8034 extending from the fluid opening 8010 to the first main channel 8026. As shown in FIG. 8A, the fluid opening 8010 may be disposed on the peripheral outer portion 8020 and/or the second upper surface 8018 of the card 8002. The fluid connection channel 8034 may be configured to introduce or allow fluid to flow into the channel system 8006 or the manifold channel assembly 8024 of the channel system. For example, as detailed in the present invention, the card 8002 may be sealed between a pouch or two layers of film (see, for example, FIGS. 8C and 8D).

Thus, in some embodiments, the connection channel 8034 may be used to fluidly transfer a liquid sample from the pouch to the array 8001 or wells 8004 of the array. In some embodiments, fluid connection channel 8034 may be in fluid communication with a fluid source (see, eg, FIGS. 8C and 8D ). In at least one embodiment, the fluid source is in fluid communication with a blister of a film pouch (see, for example, blister 566 of pouch 510 in FIG. 1 ). An optional fluid channel (see, for example, channel 565 in FIG. 1) may extend between the connection channel 8034 and the fluid source. In addition, other suitable fluid sources known in the art may be used in the present invention. Thus, each well 8004 is in fluid communication with the connection channel 8032, the connection channel 8032 is in fluid communication with the branch channel 8030, and the branch channel 8030 is in fluid communication with the first main channel 8026. And in fluid communication with the second main channel 8028. The first main channel 9026 is in fluid communication with the fluid connection channel 8034, and the connection channel 8034 is in fluid communication with the fluid opening 8010.

Thus, each well 8004 is in at least two directions ((i) directly through the first main channel 8026, its branching channel 8030 and its connecting channel 8032, or (ii) the first main channel 8026). The fluid opening 8010 from the channel 8026, indirectly through the connection channel 8032 from another row, the second main channel 8028, its branching channel 8030 and its connection channel 8032, and In fluid communication (by channel system 8006). This parallel arrangement with multiple connecting channels extending between two or more main channels creates a vacuum in all wells and allows all wells to be filled even if one of the channels is blocked. The second main channel 8028 is in fluid communication with the port channel 8036, and the port channel 8036 is in fluid communication with the vacuum port 8008. Thus, each well 8004 (by channel system 8006) is in fluid communication with a vacuum port 8008. The above structure is exemplary only, and many alternative arrangements may be used.

Card 8002 may also have one or more optional alignment indicators 8038. As shown in FIG. 8A, the optional alignment indicator 8038 may include an opening or hole extending to or through one or more surfaces of the card layer 8002. Other optional alignment indicators 8038 may include recesses (or indentations) or markings (eg, printed patterns or images) disposed on one or more surfaces of card layer 8002.

In some embodiments, one or more features or components of card 8002 (e.g., well 8004, portion of channel system 8006, port hole 8008a, alignment indicator 8038, etc.) may include the card ( 8002) It may be formed during manufacturing, for example, during injection molding of the card 8002. Alternatively, one or more features or components of card 8002 may be formed by removing material from card 8002.

8C shows an exemplary embodiment of the array assembly 8000a. The array assembly 8000a may include a card (or card layer) 8002 disposed in the pouch 8040. The pouch 8040 may include a first layer and a second layer, or may be formed of a first layer and a second layer. An exemplary card layer 8002 may be placed (eg, sandwiched) between the first film layer 8042 and the second film layer 8044. In some embodiments, the film layers 8042 and 8044 may be bonded to the upper and lower surfaces. Film layers 8042 and 8044 may also be selectively bonded to each other, for example to create fluid channels and blisters between the film layers.

Accordingly, some embodiments of the present invention include a card layer 8002 having a plurality of wells 8004 formed therein, a first film layer 8042 bonded to the first surface or surface 8016 of the card layer 8002, and /Or may be associated with an array assembly 8000a including a second film layer 8044 bonded to the second surface or surface 8018 of the card layer 8002. Although the film layer is used in the above and other embodiments, other materials may be used consistent with the application of the array assembly 8000a.

As shown in FIG. 8D, the pouch 8040 may be preformed (eg, may be preformed prior to placing the card 8002 in some embodiments).

Illustratively, the first film layer 8042 (part of) and the second film layer 8044 (part of) may be bonded to each other. First and second film layers 8042 and 8044 by hot rolling (e.g., by rolling the layers between hot rollers during manufacture), for example by thermoforming (e.g. laser welding or thermal welding) May be bonded to each other by partially bonding) or by an adhesive (eg, a temperature sensitive adhesive, a pressure sensitive adhesive, etc.). For example, the pouch 8040 may include a first bonded portion 8056a, a second bonded portion 8056b, and a third bonded portion 8056c.

Bonded portions 8056a, 8056b, and 8056c include a fluid reservoir (or blister) 8046, a fluid channel 8048 in fluid communication with the blister 8046, and a pocket 8050 in fluid communication with the fluid channel 8048. ) Can be at least partially bonded or formed. In some embodiments, the bonded portions 8056a, 8056b, and 8056c of the pouch 8040, or the first film layer 8042 and the second film layer 8044 may be (permanently) bonded to each other (e.g. For example, by heat welding or pressure sensitive adhesive). However, the blister 8046, fluid channel 8048 and pocket 8050 may be unbonded, or may be bonded in an open state (ie, reversible). In some embodiments, the unbonded portion may be defined by one or more bonding lines or welding lines 8052.

For example, the second film layer 8044 may be overlaid on the first film layer 8202 to form a lamination or lamination form. Then, at least one portion of the superimposed film layer or lamination is bonded by thermoforming (e.g., thermal welding) or an adhesive (e.g., thermal adhesive, pressure sensitive adhesive, etc.) in the joined portions 8056a, 8056b and 8056c They can be bonded together. For example, one or more heated pressure plates or other optional bonding members having a pattern of bonded portions 8056a, 8056b, and 8056c may be applied to the laminated film layer by applying heat and force (e.g., pressure) to the one or more lamination surfaces. It can be used to form pouch 8040 from 8042 and 8044.

Illustratively, as described above with respect to pouch 510, film layers 8042 and 8044 are softened so that the layers or surfaces of the layers are bonded to each other (e.g., thermoformed, or weld-welded). And/or (partially) meltable plastic or other material. For example, the first and second film layers 8042 and 8044 may include or be formed from one or more materials having similar melting temperatures (Tm), glass transition temperatures (Tg), and the like. Illustratively, the first and second film layers 8042 and 8044 may comprise or be formed from other materials including one or more polymers (eg, plastics) or combinations or mixtures thereof. Thus, the lamination or lamination of the first film layer 8042 and the second film layer 8044 may be heated with at least one Tm or Tg of one or more materials (e.g., the Tm or the material with the highest Tg). , The layer or the surface of the layer can be bonded (eg, heat-welded) together (by applying a force (eg, pressure) to the heated or molten layer).

In one or more alternative embodiments, an adhesive layer having a pattern of bonded portions 8056a, 8056b, and 8056c may be disposed between the laminated or laminated film layers 8042 and 8044. For example, the adhesive layer may be or include a pressure-sensitive adhesive (PSA).

One or more pressure plates or other optional bonding members having a pattern of bonded portions 8056a, 8056b and 8056c are applied from the laminated film layers 8042 and 8044 by applying a force (e.g., pressure) to one or more sides of the lamination. It can be used to form the pouch 8040. Alternatively, a thermosensitive adhesive or other adhesive known in the art may be used to form the pouch 8040.

As further shown in FIG. 8D, the pocket 8050 includes an unbonded open end or opening 8054 (e.g., between the bonding lines 8052 of each of the bonded portions 8056b and 8056c) and one or more. It may have an optional alignment indicator 8038a.

Optional alignment indicator 8038a may include an opening or hole through one or more of the film layers 8202 and 8044. Alternatively, optional alignment indicator 8038a may include on one or more film layers 8042 and 8044. It may include a marking (for example, a printed pattern or image) placed on it. In some embodiments, the pouch 8040 or the one or more film layers 8042 and 8044 of the pouch may include a piercing 8008b.

8C and 8D, the card layer 8002 may be inserted into the pouch 8050 through the opening 8054. The optional alignment indicator 8038 of the card layer 8002 may be aligned with the optional indicator 8038a of the first film layer 8042 and/or the second film layer 8044. May be with, thereby indicating the proper alignment and/or location of the card layer 8002 within the pocket 8050.

The film layers 8042 and 8044 may be bonded to the inserted card layer 8002, for example by thermoforming or thermal welding. For example, one or more heated pressure plates or other bonding members may bond the film layers 8042 and 8044 to the card layer 8002. The heated pressure plate may have a substantially flat and/or uniform bonding surface. Alternatively, the heated pressure plate may have a pattern of the card layer 8002 or a bonding surface having the constituent elements of the card layer.

In addition, one or more adhesive layers or components thereof (see FIG. 8A) having a pattern of the card layer 8002 are disposed on or alternatively on the counterparts of one or more surfaces 8016 and 8018 or film layers 8042 and 8044. Can be placed. As shown in FIG. 5C, for example, the card layer 5094 may be bonded to the lower film layer 5098 by the first adhesive layer 5096, and the upper film layer 5098 by the second adhesive layer 5092. 5090).

Similarly, referring to FIG. 8C, the first (lower) surface 8016 of the card layer 8002 may be bonded to the first film layer 8042 by a first adhesive layer (not shown). In this way, the second (upper) surface 8018 of the card layer 8002 may be bonded to the second film layer 8044 by a second adhesive layer (not shown). The first and/or second adhesive layer may be or comprise one or more of a pressure sensitive adhesive, a temperature sensitive adhesive, or other adhesive known in the art or described herein.

The fourth joined portion 8056d may be formed in a manner similar to that described above, through which it is possible to seal the opening 8054 of the closed structure indicated at the closed end 8054a. Accordingly, the card layer 8002 may be bonded to the pouch 8040 or the film layers 8042 and 8044 of the pouch, and may be sealed within the pocket 8050.

In some embodiments, the non-bonded pocket portion 8050a may be disposed on one or more sides of the card layer 8002. As shown in FIG. 8C, for example, the non-bonded pocket portion 8050a may surround the card layer 8002 inside the sealed pouch 8040. The unbonded pocket portion 8050a may be defined by a bonding line (eg, welding line) 8052 of the bonded portions 8056a, 8056b, 8056c and 8056d. Similarly, fluid channel 8048 and reservoir (or blister) 8046 can be defined by bonding line 8052 of bonded portion 8056a.

In an alternative embodiment, the array assembly 8000a may be formed of a card layer 8002 disposed between the unbonded sheets 8202 and 8044. Exemplarily, the lamination or lamination of the card layer 8002 disposed between the first film layer 8042 and the second film layer 8044 is as described above and/or the pattern of the pouch 8040 or the pouch. It may be bonded with a bonding line 8052. For example, the first and second film layers 8042 and 8044, and the card layer 8002 comprise one or more (respective) materials with similar melting temperatures (Tm), glass transition temperatures (Tg), etc. It may be made of the above material.

Illustratively, the first and second film layers 8042 and 8044, and the card layer 8002 comprise or include other materials including one or more polymers (e.g. plastics) or combinations or mixtures thereof. Can be made of. In a preferred embodiment, the first and second film layers 8042 and 8044, and the card layer 8002 may include or consist of polypropylene or a mixture thereof. However, the layer can also be formed of other materials known in the art.

The lamination or lamination of the card layer 8002 disposed between the first film layer 8042 and the second film layer 8044 is at least Tm of one or more materials (e.g., the material with the highest Tm or Tg) or Can be heated to Tg. Forces (eg, pressure) can also be applied to the stratification, through which the layer or the surfaces of the layers can be bonded to each other (eg, thermally welded).

Alternatively, as described above, one or more adhesive layers (e.g., an adhesive layer having a pattern of the card layer 8002 and/or a bonding line 8052 of the pouch 8040) is the card layer 8002 and one or more first It may be disposed between the film layer 8042 and the second film. Through this, the card layer 8002 may be bonded to the first film layer 8042 and/or the second film layer 8044 by an adhesive layer.

As further shown in FIG. 8C, a fluid or liquid (sample) 8062 may be placed in a fluid reservoir (or blister) 8046.

In the illustrated embodiment, before inserting the card layer 8002 into the pocket 8050, the fluid 8062 may be first placed (eg, injected) into the blister 8046. One or more additional fluid channels may be connected and/or in fluid communication with a fluid reservoir (or blister), as shown in the corresponding figures (see, eg, FIGS. 1 and 3) in another embodiment. Accordingly, one or more additional fluid channels 8048 may extend from blister 8046 through which fluid 8062 may be introduced into blister 8046 after formation of pouch 8040.

As in region 8060 of fluid channel 8048. An open seal may also be formed in the pouch 8040. As described above, the open seal is a flexible seal, or an open seal formed by static electricity, contact, or other attraction between the film layers 8042 and 8044, rather than by thermoforming or bonding through an adhesive (e.g., peelable Seal) or may include it. The open seal may be configured to hold fluid within blister 8046 (through this, for example, fluid 8062 may not unintentionally and/or prematurely flow into pocket 8050).

The array assembly 8000a or the pouch 8040 thereof may include at least one opening or through portion 8008b. For example, the through portion 8008b may be formed in one or more of the film layers 8062 and 8044 during the manufacture of the pouch 8040 or the film layers 8042 and 8044 thereof. Alternatively, while performing the exemplary analysis method, the through portion 8008b may be formed (in the array assembly 8000a). For example, the through part 8008b may be formed by a through element of an analysis device (not shown) before the vacuum supplied from the vacuum port 8008 is applied. For example, the through portion 8008b may be substantially aligned with the hole 8008a of the card layer 8002. In some embodiments, the through portion 8008b and the hole 8008a may form the vacuum port 8008. That is, the vacuum port 8008 may include a through portion 8008b and a hole 8008a in some embodiments.

8A and 8B, by means of a sealing channel system 8006 (e.g., at or near a fluid opening 8010, such as an area 8060 of an adjacent fluid channel 8048). In other words, the fluid channel has a pressure seal or an open seal (e.g., a soft seal or a peelable seal) between the film layers 8042 and 8044 forming the fluid channel 8048, an array assembly ( The 8000, 8000a can be evacuated by applying a vacuum to the vacuum port 8008 by a vacuum device (eg, a piston pump (not shown)).

The sealing in region 8060 is that the applied vacuum is applied to the port channel 8036, the second main channel 8028, the respective branch channels 8030, the first main channel 8026, the fluid connection channel 8034, respectively. The connection channel 8032 and each well 8004 of the array 8001 may be exhausted. In this embodiment, the channel system 8006 may include or consist of a vacuum channel. For example, since vacuum is applied to the channel system prior to filling the array assembly 8000,8000a (e.g., fluid sample 8062), the fluid sample (or the first stage amplification of a two-stage PCR system) Is not introduced into the vacuum device. As a result, contamination caused by being connected to a vacuum device can be minimized or prevented.

With the array assembly 8000 evacuated and the applied vacuum maintained (e.g., by sealing the vacuum port 8008 in the region 8064 of the port channel 8036, for example, to pressure and/or heat sealing). By), the sealing portion of the region 6060 can be opened. Fluid into the array assembly 8000 (i.e., through the fluid opening 8010, through the first main channel 8026, through the branch channel 8030, through the connection channel 8032, the array 8001) And into the well 8004). By sealing the port channel 8036 in the region 8064, fluid also enters the second main channel 8028 and a portion of the port channel 8036 (to the seal). In this embodiment, the channel system 8006 may include or consist of an array fill channel. Accordingly, the channel system 8006 may include or consist of a vacuum channel (or channel assembly) and an array fill channel (or channel assembly).

In the illustrated embodiment, each well 8004 may be separated from all other wells 8004 by sealing a branch channel 8030 between the connection channels 8202. In at least one embodiment, the plurality of seals may extend across one or more (eg, all) branch channels 8030 between one or more connection channels 8032. Since each of the wells 8004 of a particular row 8022 is connected to an adjacent branch channel 8030 via a specific connection channel 8032, across the branch channel 8030 between the connection channels 8032 (e.g., The length of the branch channels 8030 and perpendicular to the rows 8222 of the wells 8004) can be sealed, which can significantly reduce or eliminate the crossover between the wells 8004.

By using the channel system 8006 as a vacuum channel and an array fill channel (manifold type), the card 8002 can be kept relatively small. However, in some embodiments, the array assembly 8000 may include a vacuum channel and a separate array fill channel. For example, in an alternative embodiment, the array assembly 8000 may include a dual manifold configuration that is not in fluid communication with the first main channel 8026 and the second main channel 8028. Instead, the first branched (filled) channel set 8030 may extend from the first main channel 8026, and the second branched (vacuum) channel set 8030 is the second main channel 8028. Can be extended from

Each connection channel 8032 may extend from each branch channel 8030 to each well 8004. The first connection channel 8032 may be fluidly connected to a specific well 8004 adjacent to the branched vacuum channel 8030 on the first side of the well 8004, and the second connection channel 8032 is a well 8004 ) May be fluidly connected to a specific well 8004 adjacent to the branched fill channel 8030 on the second side of the ).

As mentioned above, the vacuum port 8008 need not be disposed on the opposite side of the well 8004 from the fluid opening 8010. For example, the vacuum port 8008 may be disposed on a surface of the well 8004 that is the same as or adjacent to the fluid opening 8010. For example, the vacuum port 8008 and the fluid opening 8010 may be disposed at the opposite end of the same or adjacent side of the well 8004.

The vacuum port 8008 may be disposed adjacent to the fluid opening 8010. For example, in an alternative embodiment, the channel system 8006 or manifold channel assembly 8024 thereof may have only one main channel 8026. Illustratively, the port channel 8036 may extend from the main channel, through which the vacuum port 8008 or a hole 8008a thereof may be in fluid communication with the main channel 8026. In this configuration, the fluid channel in fluid communication with the connection channel 8034 can be resealably sealed, and a vacuum can still be applied through the vacuum port 8008.

9A and 9B show an exemplary embodiment of a second step 9558 using a barrier layer. The structure sandwiched between the first layer 9518 and the second layer 9519 of the pouch 9510 forms a high density array 9801 having wells 9852.

As best shown in FIG. 9B, the through-part layer 9585 having through-parts 9586 is a high-density array 9801 as a physical barrier for minimizing the cross-action between the wells 9852 when the array 9601 is filled. It is provided on one side of. Further, a second layer 9587 is provided on the opposite side of the high density array 9801 to form the bottom of the well 9582. In the illustrated embodiment, the second-stage array 9580 is manufactured to have the through-part layer 9585 bonded to the first layer 9601.

In layer 9518, a feature is formed, for example, by bonding selected regions and impressing the feature into the film layer 9518. This forms a fluid flow channel 9053 and a coexisting vacuum channel 9050 to create a vacuum in the array in situ (ie, point of use) as an alternative to fabricating and storing the array under vacuum conditions.

Illustratively, layer 9518 may include a fluid fill channel 9053 formed by bonding layer 9518 to layer 9585 (eg, laser welding). A fluid fill channel 9053 defined by laser welding line 9042 is formed in fluid communication with each of the through holes 9586 in the layer 9585. The vacuum passage 9050 is formed at the same time as the fluid filling channel 9053, and may be in fluid communication with the through hole 9586 of the layer 9585. In an embodiment, the vacuum passage 9050 may be thermoformed by forming a film shape of the film layer 9601 with a hot wire or the like to impress the channel with a film.

Heat may be formed by forming the film of the film layer 9518 with a hot wire and injecting the film into the channel. Illustratively, the through layer 9585 and the first layer 9518 are bonded (eg, welded) to each other around the outer shell of 9026. The through layer 9585 is bonded to the high-density array layer 9601 by, for example, an adhesive layer and/or heat sealing, and the first layer 9518 is of the through-layer hole 9586 for defining the fill channel 9053. It is bonded (eg, laser welded) to the through-part layer 9585 of the plurality of welding lines 9052 parallel to the row. The fluid filling channel 9053, the through layer 9585 and the opening 9586, and the array well 9582 may be in fluid communication with the upward fluid well through the fluid channel opening 9565.

9C shows a cross-sectional view along line C-C of the layers of array 9580. While FIG. 9A is shown in an exploded view, FIG. 9C shows the relationship between the pouch film and the array layer, the vacuum passage 9050, and the fluid flow channel 9053. The "stacking" of the second stage amplification region 9558 is a first pouch film layer 9518 (i.e., a first outer layer of a pouch), a penetrating portion layer 9585, an array 9558, a second layer 9587 (ie , An array backing layer) and a second pouch film layer 9519 (ie, a second outer pouch film layer).

The cross section of FIG. 9C shows one through 9586 and one well 9582 and how they relate to vacuum passage 9050, fluid flow channel 9053 and welds 9022 and 9026. One through portion 9586 and one well 9852 are shown for ease and clarity, and as shown in Figs. 9A and 9C, the vacuum passage 9050 and the fluid flow channel 9053 are a second stage amplification. In fluid communication with all of the penetrations 9586 and wells 9582 of the region 9580.

In the illustrated embodiment, the fluid flow channel 9053 comprises a first layer 9518 and a penetration layer (e.g., laser welded) bonded to each other (e.g., laser welded) at welds 9052 and 9026 defining the fluid flow channel 9053. 9585) is formed as an open space between.

Illustratively, the vacuum passage 9050 may be formed as a sub-channel of the layer 9518. In the illustrated embodiment, the vacuum passage 9050 may be of other shapes, but the channel 9053 is kept open and the channel 9053 is connected to the through portion 9586 and the well 9582 of the through layer 9585. It can have an arch shape designed to connect. In the absence of a vacuum passage 9050 formed, the channel 9053, along with a specific film or other material, can be "kissed" closed when vacuum is applied to the channel to prevent evacuation of the array. In one embodiment, the vacuum channel 9050 may be formed in the layer 9518 having a heat-forming fixture (eg, a hot'debossed' wire of an appropriate shape). In another embodiment, the vacuum channel 9050 may be formed by laser etching, gyrography, or the like.

The through layer 9585 and the second layer 9587 may be plastic films sealed into the high density array 9801 by, for example, heat sealing (e.g., thermally active sealing), but other sealing methods may be used. . In addition, the material used for the high-density array 9581 and the material used for the penetration layer 9585 and the second layer 9587 may be compatible with each other in a sealing method and a chemical substance used.

When used for PCR, examples of compatible plastics that can be used in the high density array 9801 and that can be heat sealed are PE, PP, Monprene® and other block copolymer elastomers. If a fluorescent dye is used for detection chemistry, in order to minimize signal bleeding from one well 9582 to a well surrounding it, and to make at least one of the layers 9585 and 9587 relatively fluorescently transparent. For this purpose, it may be desirable for the high-density array 9801 to be formed of black or other material that is relatively fluorescently opaque.

For the through layer 9585 and the second layer 9587, a steel engineering plastic laminate such as Mylar® or PET with a heat sealable plastic layer such as PE, PP and Dupont Surlyn® may be used. For adhesive-based systems, rigid engineering plastics such as PET or polycarbonate can be used to form a high-density array (9581), and a PCR-compatible plastic film can be used as the penetration layer (9585) and the second layer (9587). I can. In one exemplary embodiment, the high density array 9801 is formed of black PE, and the composite polyethylene/PET laminate (or Xerox®PN 104702 hot laminating pouch material) is heat-sealed with a high density array 9801 through layer 9585 ) And the second layer 9587, and the composite polypropylene/PET is used for the first and second layers 9518 and 9519 of the pouch 9510.

The through part 9586 may be aligned with the well 9582. The penetrating portion 9586 may be small enough so that fluid does not easily flow through the penetrating portion 9586 without a slight force. For example, the penetration portion may be 0.001-0.1mm, more specifically, 0.005-0.02mm, and even more specifically, about 0.01mm.

In an exemplary embodiment, by forming a vacuum on the well 9582 through the vacuum channel 9050 through the vacuum port 9501 and then sealing (e.g., heat sealing) the vacuum channel 9050, in real time ( Insitu), a vacuum may be applied to the second stage amplification region 9658.

Substantially, when a fluid is provided, the vacuum moves the fluid through the penetrations 9586 to each well 9582. When the well 9582 is filled and the pressure is equilibrated, there is no more force to move the fluid into or out of the well 9582. Illustratively, the well may be sealed after filling by applying a heat seal that is substantially perpendicular to the fill channel 9052. In another embodiment, a bleeder (not shown, disposed similarly to the bladder 880/882) adjacent to the second amplification region 9580 is activated to press the first layer 9518 against the high density array 9801 And sealing the fluid inside the well 9582. The first layer 9518 of the pouch 9510 is used to seal the well 9582, but an optional sealing layer may be provided between the through layer 9585 and the first layer 9510.

In one illustrative example, the second stage amplification region 9558 may be prepared as follows.

The high-density array 9801 may be fabricated by punching, molding, or other methods of forming an array of wells 9852 in a plastic sheet (eg, 0.1 to 1 mm thick). The wells can form any regular or irregular array desired, and may have a volume of, for example, 0.05 μl to 20 μl, more preferably 0.1 μl to 4 μl.

A backing layer (e.g., 9587) is then laminated to the first surface 9601a of the high-density array 9801, for example by heat or adhesive. As shown in FIG. 9B, a second layer 9587 is applied to the first surface 9801a.

Subsequently, reagent 9589, for example, a unique array of chemical constituents, such as PCR primer pairs, can be manually or automatically pipetted (e.g. pin-spotter, dot matrix printer, small automatic pipette or microfluidic microfluidic An x/y positionable spotter, such as a contact spotter, can be placed in the well automatically) using, for example, the spotter described in US Patent Application No. 2017-0209844, which is incorporated herein by reference. After the reagents 9589 are dried in each well 9852, a penetration layer 9585 pre-bonded to the layer 9518 may be applied to the second surface 9801b of the array 9801.

The array 9801 may be bonded to the layer 9519 of the pouch 510 and sealed in place. Illustratively, it may be bonded and sealed by heat sealing using an adhesive, ultrasonic welding, mechanical closure, or other method surrounding the array 9801 inside the pouch 510 in the blister 580.

The second stage reaction zone 9558 may be in fluid communication with a downward fluid blister (e.g., a first stage reaction zone) through a channel 9565, and the liquid flows from the channel 9565 to the second stage. It may flow into the reaction region 9580 and through the channel 9565 through the penetrating portion 9586, and the channel 9565 may be in fluid communication with the fluid flow channel 9053 and the vacuum passage 9050. .

The second-stage reaction region 9558 may be used in a manner similar to the second-stage array 11000 of FIG. 7A or the array assembly 8000a of FIG. 7A. Since a vacuum can be applied to the array 9801, the liquid sample is transferred from the downstream fluid reservoir, blister to the second stage amplification region 9558, the liquid sample in the fluid flow channel 9053 and through 9586. It may be introduced into the well 9852 through.

Since the array assembly is provided with a vacuum port in fluid communication with the plurality of wells, vacuum can be applied (locally) to the array assembly (rather than during manufacture or assembly of an assay device such as a pouch in which the array assembly is placed). have.

Therefore, the evacuation chamber does not require that the product be assembled in a vacuum state, and thus no vacuum needs to be applied during manufacture. Instead, a piston pump (e.g., a syringe or syringe-type vacuum device) or other vacuum device may be used to apply the on-demand vacuum, rather than at the point of use and during assembly of an assay device such as a pouch in which the array assembly is placed. It can be used to apply a vacuum during the analysis of other stages of the analysis method. Thus, the evacuation chamber does not need to apply a vacuum, so that the product can be assembled under vacuum conditions. Instead, a piston pump or other vacuum device can be used to apply the vacuum.

In one exemplary fabrication process, the volume of the evacuation chamber used during fabrication of an assay device comprising an array assembly packaged under vacuum may be several times larger than the volume of the well and channel system of the array assembly.

Venting a larger volume may require a larger pump and/or may take longer than venting a smaller volume. Larger pumps, such as diaphragm vacuums or rotary vane pumps, may not perform as well at forming (hard) vacuums with larger volumes than piston pumps, which generally create (hard) vacuums with smaller volumes.

In addition, larger pumps capable of increasing the vacuum level created by the piston pump can be much more expensive to acquire, operate and/or maintain than the piston pump. In addition, a single stock of piston pumps can create a vacuum, and in larger, diaphragm or rotary vane pumps more time is spent creating the vacuum. Thus, within a given time, the piston pump can build a more complete (eg, closer to the final value) vacuum than a larger diaphragm or rotary vane pump.

As mentioned above, vacuum can also be applied on demand, such as at the point of use. Thus, the vacuum need not be maintained for a long period of time by the array assembly. Instead, unlike conventional systems in which the assay device is packaged in a vacuum state, the assay device must maintain the vacuum during storage, transportation, transfer to the assay device, and preliminary stages of the assay process, while on-demand vacuum opens the fluid seal to allow the fluid to drain. It may be applied and/or maintained at the immediately preceding well moment until the vacuum is released or used to enter (eg, through a channel system).

For example, in a preferred embodiment, before the liquid sample enters the well, the vacuum needs to be held for several seconds by the array assembly (ie, long enough to create a vacuum seal and open the fluid seal).

In this way, almost the entire level of vacuum formed can be used to release the vacuum to introduce a complete and constant amount of fluid into each well when opening the fluid seal. Further, since the vacuum does not enter through the liquid sample through the fluid opening, the level or strength of the vacuum is not limited to the partial pressure of the fluid sample. This stronger vacuum level of decompression (e.g., between 2 mBar and 140 mBar) completely evacuates air from the array assembly so that residual air in the array assembly is minimized, trapped by fluids entering the channels and wells or array assembly. Minimize possible air bubbles.

In addition, the array assembly can be structured (eg, size and structure) so that it does not need an exhaust chamber to apply a vacuum. Instead, the bonding volume of the well and channel system can be small enough to allow nearly all air to be expelled into a single stock of the piston pump.

11A schematically depicts a system 1100 that includes an instrument 1101 capable of forming a vacuum in a reaction vessel in situ while performing one or more analytical method steps. An exemplary reaction vessel configured to apply vacuum to one or more portions of the reaction vessel (eg, blister, second stage array, reagent well or blister, etc.) is shown at 1102. A reaction vessel 1102 having a specific shape and layout is provided, but this is merely exemplary and other reaction vessels (eg, pouch 8000a) may be suitable for in situ applications of vacuum.

The reaction vessel 1102 includes a sample blister 1103 that can be used to introduce a sample into the reaction vessel. In one embodiment, the sample may be introduced with a cotton swab 1104. However, the cotton swab 1104 is only an example. In other embodiments, a liquid sample may be introduced with, for example, a transfer pipet, and a dry sample (eg, a contaminated piece) may be placed in the sample blister 1103. The reaction vessel 1102 further includes a decomposition zone 1105.

In the decomposition region, cells and viruses in the sample can be degraded to free nucleic acids for analysis, and in the reaction region 1106, the first step for nucleic acid recovery and purification, a first step multi-complex PCR, and a second step PCR Various method steps may be performed, such as the preparation of step products, but are not limited thereto.

In one embodiment, the decomposition region 1105 and the reaction region 1106 may be conjugated into one region for decomposition, purification of nucleic acids, and first-step PCR. In other embodiments, the decomposition region 1105 and the reaction region 1106 may be divided into a number of individual blisters in which individual functions are performed. Pouch 510 may be an example of such a reaction vessel having a number of individual blisters in which different functions are performed.

Exemplary reaction vessel 1102 may further include a number of reagent blisters 1107a-107d that can be used to provide reagents for analysis performed in the reaction vessel. Reagent blisters may contain dry reagents, liquid reagents, or a combination of both. Although four reagent blisters are shown, more or less blisters may be included depending on the analysis performed in the reaction vessel. As such, the reagent blisters can be linked to the reaction blisters in a number of different structures, owing to the analysis performed in the reaction vessel.

The reaction vessel 1102 may further include a number of channels 1109a-1109g used to connect with regions of the reaction vessel. In one embodiment, the channels 1109a-1109g may be openably sealed as described in detail in another sealing example of the present invention. The reaction vessel 1102 may further include an array 1181 comprising a number of wells 1181 that may be configured for individual reaction analysis.

Although the apparatus 1101 is shown schematically only, those skilled in the art know that the apparatus 1101 may be configured to perform a number of operations on the reaction vessel 1102, heat treatment steps, cooling steps, etc. in the course of performing the analysis method. I will understand.

In one embodiment, the device 1101 may include one or more heaters configured to control the temperature of one or more portions of the reaction vessel 1102. For example, such a heater may be configured to thermally cycle one or more portions of the reaction vessel 1102 for one or more PCR reactions.

Similarly, such heaters may be configured for one or more isothermal processes in one or more portions of reaction vessel 1102. In one embodiment, the device 1101 is a disassembly device (e.g., a bead beater such as component 804 of FIG. 2, a paddle beater, ultrasonic wave) for performing cell dissolution in one or more portions of the reaction vessel 1102 Processor).

In one embodiment, the instrument 1101 may include a magnet for magnetic bead capture, and the instrument 1101 may include an actuator for moving fluid within one or more portions of the reaction vessel 1102. In one embodiment, the device 1101 includes one or more seals (e.g., shrinkable seals, and/or heat seals) for controlling the movement of fluid within one or more portions of the reaction vessel 1102. can do. In one embodiment, instrument 1101 may be a light source and image capture system for optical (eg, fluorescence) excitation and data collection from one or more portions of reaction vessel 1102.

In addition, the instrument 1101 may be a computer (internal or external computer or both), an integrated display, a heat seal device for applying heat seal to one or more portions of the reaction vessel 1102 and/or components of the instrument 1101 A compression member (eg, an expandable bladder) for compressing one or more portions of the reaction vessel 1102 against. For example, a compression member may be used to improve thermal contact between one or more portions of the semi-container and one or more heaters.

In one embodiment, instrument 100 1 is configured to perform at least one PCR reaction in a reaction vessel. Other components that may be included in the instrument 1102 are shown in Figures 2-4 and described herein. The shape and arrangement of the components shown in FIGS. 2 to 4 may be different in the device 1102, but the functions may be the same or similar.

In addition to the foregoing, the apparatus 1101 and the reaction vessel 1102 may be configured to create a vacuum in one or more portions of the reaction vessel 1102 prior to or during performing the analytical method. As detailed in other embodiments of the present invention, having one or more portions of reaction vessel 1102 under partial vacuum may facilitate filling one or more portions with fluid. For example, filling the array well 1182 can be significantly facilitated by having the array 1181 under partial vacuum.

In one embodiment, the reaction vessel 1102 may include a channel system similar to that shown in FIGS. 7A, 8A, 8C and 9A, for example to apply a vacuum to one or more portions of the reaction vessel. In addition, the instrument 1101 may include a system for forming a vacuum in one or more portions of the reaction vessel 1102 during performing the analytical method. The vacuum system can include a vacuum line 1110 and a vacuum source 1112. Vacuum source 1112 can be essentially any vacuum source or vacuum generating device known in the art. Examples include, but are not limited to, piston pumps (e.g., syringe pumps), positive displacement pumps, momentum transfer pumps (also referred to as molecular pumps) and capture pumps (also referred to as molecular traps). In one embodiment, vacuum source 1112 may include an evacuated chamber connected to a vacuum pump.

In such a system, a partial vacuum in the evacuation chamber may be used to create a vacuum in one or more portions of the reaction vessel 1102 and, in turn, a vacuum pump may be used to create or maintain a vacuum in the evacuation chamber. In one embodiment, vacuum source 1112 and vacuum line 1110 may form a vacuum in array 1181 at vacuum port 1108. In one embodiment, vacuum source 1112 may be fluidly bonded to at least one additional vacuum line to form a vacuum in at least one additional portion of reaction vessel 1102. For example, FIG. 11A shows an extension 1118 in vacuum line 1110 that can be used to create a vacuum in at least one reagent blister through port 1116.

In one embodiment, the instrument 1101 may be used to apply a vacuum to one or more portions of the reaction vessel 1102 while performing the analysis method or after the instrument has performed at least one step of the analysis method. For example, the device 1101 may be one or more method steps or steps in a first reaction zone (e.g., reaction zone 1105) during or after insertion of the reaction vessel into the device, or during or after hydration of the sample. One or more portions of the reaction vessel 1102 with fluid during or after carrying out the reaction, or during or after carrying out one or more method steps or reactions in a second reaction zone (e.g., reaction zone 1106) In preparation for filling, a vacuum can be formed in the reaction vessel 1102. For example, the instrument 1101 may create a vacuum in preparation for filling fluid in the array 1181 or reagent blister 1107d (or other reagent blister).

In one embodiment, one or more method steps or reactions in the first reaction zone include sample decomposition (e.g., by bead beating), separation of decomposition particles from decomposition products, decomposition products into other chambers of the reaction vessel. The step of moving, mixing the silica magnetic beads with the decomposition product; Or capturing the magnetic beads and moving them to another chamber of the reaction vessel, but is not limited thereto.

In one embodiment, one or more method steps or reactions in the second reaction zone include mixing magnetic silica beads with the degradation product from the first reaction vessel, the degradation product used after the capture of the nucleic acid into another chamber of the reaction vessel (e.g. For example, moving the residual decomposition product), performing washing of the magnetic beads at least once, moving the washing solution to another chamber of the reaction vessel, eluting nucleic acids from the magnetic silica beads, the first PCR reaction ( For example, single PCR, complex PCR), or preparation for performing a second PCR reaction in the wells of the array, may include, but are not limited to, diluting the product of the first PCR reaction. Does not.

11B and 11C show in more detail a system for creating a vacuum in an array and reagent blister. FIG. 11B shows vacuum manifolds 1128 and 1130 for array 1181. Details of embodiments of vacuum manifolds and methods relating to arrays, array wells, etc. are shown in FIGS. 8A-8C, and are used herein. Is explained. In the illustrated embodiment, vacuum line 1110 and vacuum source 1112 may be connected to vacuum port 1108 through vacuum hub 1111. The vacuum hub 1111 may be an elastic member such as a suction cup or nipple known in the art.

In one embodiment, the vacuum hub 1111 may include a sharply shaped member (eg, a needle, etc.) positioned to penetrate the film layer through the vacuum port 1108. In another embodiment, the vacuum port 1108 may include a flapper valve or other valve, whereby the vacuum port may be normally sealed from the outside. However, the valve may be opened to allow air to escape when a vacuum is formed in the vacuum port 1108. After a vacuum is formed in the array 1181, a seal (eg, a heat seal) may be applied in the area of 1184 to seal the channels 1128 and 1130 from outside air.

11C shows an exemplary blister 1107d that may be configured to have a vacuum formed in situ. Although one reagent blister 1107d is shown here, multiple reagent blisters can be connected to one vacuum system, or a reaction vessel such as 1102 can be configured to have a vacuum applied in situ. It may contain two or more reagent blisters.

In the illustrated embodiment, vacuum line 1118 and vacuum source 1112 may be connected to reagent blister 1107d through vacuum port 1116, and reaction vessel vacuum line 1114 through vacuum hub 1117. Can be connected with. Like the vacuum hub 1111, the vacuum hub 1117 may be an elastic member such as a suction cup, nipple, or the like known in the art. In one embodiment, the vacuum hub 1117 may include a sharp member (eg, a needle, etc.) disposed to penetrate the film layer through the vacuum port 1116.

In another embodiment, the vacuum port 1116 may include a valve, through which the vacuum port can be normally sealed from the outside, and the valve allows air to escape when a vacuum is formed in the vacuum port. Can be opened. The reaction vessel vacuum line 1114 can be formed as described in other embodiments of the present invention to keep the line open when a vacuum is applied.

For example, the reaction vessel vacuum line 1114 may include an embossed arch formed on a film used in manufacturing the reaction vessel 1102, and the vessel vacuum line 1114 may be formed on a card material. As described in another embodiment of the present invention, the open seal 1121 in the channel 1109e prevents the application of the vacuum to the blister 1107d from the state applied to the other dudddur of the reaction vessel 1102. I can. After a vacuum is formed in the reagent blister 1107d, a seal (eg, heat seal) may be applied to the area 1122 so that the vacuum is preserved in the reagent blister 1107d after the vacuum hub 1117 is removed.

In one embodiment, reagent blister 1107d may contain dried chemicals 1120 that need to be rehydrated for use in analysis. For example, in order for the rehydration liquid to flow into the reagent blister 1107d without air blocking fluid flow so that air bubbles do not subsequently enter the reaction chamber (e.g., the reaction chamber 1106), the reagent blister ( It may be advantageous to apply a partial vacuum to 1107d).

The dried chemical substance 1120 may be air dried or lyophilized, and may include a reaction component (eg, enzyme), a buffer extract, a stabilizer, and the like. The freshly dried chemicals 1120 may be in the form of powders, dried reagent pills, reagents placed on filter paper, or other forms known in the art.

It is understood that in various embodiments of the arrays described herein, reaction components may be located in the wells. For example, primers can be placed in each well to initiate a nucleic acid amplification reaction. Each well may have a different pair of primers, and various wells may contain copies of primers placed in other wells, or any combination thereof. Additional substances may be disposed comprising one or more nucleotide triphosphates (NTPs), polymerases, magnesium or other components. When these components are placed and dried during manufacture, the components are often dried around the outer shell of the well, leaving a relatively small surface area for rehydration. As an example of fast PCR, for example, referring to PCT/US2017/18748, which is incorporated herein by reference, the time required for such rehydration may be extended through multiple PCR cycles, which may require additional cycle time. I can.

10A-10F, one method of reducing the time required for rehydration and dissolution during the second stage PCR thermal cycle is, for example, during sample preparation or during the first stage PCR, in the reaction process when the array is used. Some time before the array is introduced into the hydration solution.

10A-10E show an example of a method of rehydrating contents and initiating a reaction in an exemplary well of a second stage array. 10F shows an alternative method of initiating the reaction with the method shown in FIGS. 10A-10E.

10A-10F illustrate one well 1082 of the array 1080, but multiple wells are arranged in an array such as, for example, array 580, array 5081, and array 8000 and other array structures. There may be. An exemplary well 1082 may be formed in the array layer 1081, and a boundary may be defined by the through layer 1085 and the second layer 1087.

Layer 1018 may be the outer layer of the pouch, similar to layer 518, or layer 1018 may be an extra layer of flexibility provided for the rehydration method. Array 1080 may be used in various embodiments of the present invention, or may be used in other sample processing systems.

The array 1080 may be arranged with various components 1040 for sample processing, for example the above components for PCR. An exemplary method of placing the array 1080 is described in US Patent Application No. 2015/0283531, which is incorporated herein by reference.

However, other methods of arranging the array 1080 may be used within the scope of the present invention. After placement, the array 1080 may be dried. 10A, component 1040 may be dried at the edge of well 1082. Optionally, the array 1080 may be vented at the point of use, as shown by the arrows in FIG. 10A and described in detail in another embodiment of the present invention. As such, the array 1080 may be evacuated during manufacture and stored in a vacuum state until use.

In FIG. 10B, the hydration solution 1070, for example, water or a buffer solution, is introduced into the well 1082 through the opening 1086. The opening 1086 may be a penetrating portion such as the penetrating portion 586 described in another embodiment of the present invention, and the opening 1086 may prevent fluid from flowing easily through the opening 1082 when a slight force is not applied. It can be small enough.

However, in some embodiments, it may be desirable for the opening 1086 to be larger, or it may be desirable to use a different barrier layer. After filling with the hydration liquid 1070, the excess hydration liquid is used to roll the fluid into a waste container (not shown) using, for example, a roller 1050 as shown in FIG. 10C, or, for example, the array 1080 ) Can be removed from the array by applying pressure to the array, by means of a bladder placed on the instrument adjacent to ). Since the material of each array requires rehydration, when the excess hydration liquid 1070 is immediately removed, the reaction component 1040 starts to be rehydrated with the hydration liquid 1070, thereby minimizing the amount of cross contamination between wells. I can. Optionally, as the excess hydration liquid 1070 is used as a waste container, pressure may be applied to the layer 1087, which pressure decreases the volume of the well 1082 so that the well 1082 is under-filled (filled Not in a state).

In FIG. 10D, after the first step PCR, the first step reaction mixture 1045 may be moved into the array 1080 by, for example, pressure. Since each well is flexible and already filled but not overfilled, exemplarily due to the removal of excess hydration, each well contains a small amount of reaction material, such as the first stage PCR reaction mixture 1045. can do.

Since only a small amount of the first-stage PCR reaction mixture 1045 is introduced into each well, the dilution step of the first-stage PCR reaction mixture may be omitted before introduction of the first-stage PCR reaction mixture. If the well 1082 is under-filled (unfilled) as described above, the amount of the first-stage PCR reaction mixture 1045 that can be introduced into each well may be somewhat large, and accordingly , The amount of diluent may be slightly reduced. While removing excess hydration liquid 1070, the amount of pressure applied on layer 1087 can be adjusted to provide an appropriate amount of diluent.

As shown in FIG. 10E, roller 1050 or other pressure can be reused to remove excess first stage PCR reaction mixture 1045. In the above exemplary embodiment, only a small amount of the first step PCR reaction mixture 1045 may be pressed through the opening 1086. 10F shows an alternative embodiment, in which instead of forcing the excess first stage reaction mixture 1045 discharged from the array 1080 in that embodiment, the layer 1018 is illustratively an arrow 1090. By pressure or heat sealing at, the through layer 1085 may be sealed.

In some embodiments, the mass of the first stage PCR reaction mixture 1045 is sealed outside the opening 1086, and the first stage PCR reaction mixture 1045 and components 1040 will continue to be mixed during subsequent thermal cycling. I can. In the above embodiment, the amount of the diluent may be smaller than that of the embodiment shown in FIG. 10.

Since this pre-hydration of the array 1080 may include the relevant reactants, it may be desirable to omit one of the reactants in the array until the first stage reaction mixture is provided to the array so that the reaction mixture is incomplete, The reaction in the array may not start until the first stage reaction mixture is provided.

For example, when the second-stage array 1080 is used for PCR, magnesium may be omitted from both the dried array component and the hydration solution, thus preventing the formation of primer dimers during rehydration. In this method, high-density magnesium may be added to the first-stage reaction mixture before providing the first-stage reaction mixture to the array, and when a small amount of first-stage reaction mixture is introduced into each well, a high-density magnesium is added. It can be provided as an array to allow PCR to proceed.

Magnesium is an exemplary component, and other components can be used to control the onset of amplification. Also, a complete mixture can be provided to each well, and the start of the reaction can be controlled by controlling the reaction temperature, for example by cooling using a heater such as heater 888.

Accordingly, embodiments of the present invention can provide many advantages over conventional systems.

Although the foregoing detailed description refers to specific exemplary embodiments, the present invention may be implemented in other specific forms without departing from its spirit or essential characteristics. Accordingly, the described embodiments should be regarded as merely illustrative and non-limiting in all respects. For example, various substitutions, changes and/or modifications of the features of the invention described and/or illustrated herein, and the description of the present specification that may occur to those of ordinary skill in the art and have ownership of the invention And/or further application of the illustrated principles may be made in the described and/or illustrated embodiments without departing from the spirit and scope of the invention as defined by the appended claims.

The limitations recited in the claims are to be interpreted broadly based on the language used in the claims, and are not limited to the specific examples set forth in the foregoing detailed description. In addition, the specific examples should be construed as non-exclusive and non-exclusive. All changes that fall within the meaning and equivalent scope of the claims should be included within the scope.

It will also be appreciated that various features of certain embodiments may be compatible with, combined with, included, and/or incorporated into other embodiments of the present invention. For example, systems, methods, and/or products in accordance with certain embodiments of the invention may include features described in other embodiments disclosed and/or described herein. Accordingly, disclosure of specific features for specific embodiments of the present invention should not be construed as limiting the application or inclusion of the features to specific features. In addition, features described in various embodiments may be optional and may not be included in other embodiments of the present invention unless a feature is described as required in a specific embodiment. Further, any feature of this specification may be combined with any other feature of the same or different embodiments disclosed herein, unless a feature is described as requiring another feature in combination with the feature.

Claims (68)

delete delete delete delete delete delete delete delete delete delete delete delete delete (a) preparing a sample container including a reaction region in fluid communication with the array assembly;
(b) forming a reaction mixture by performing an analysis method on the sample in the reaction zone;
(c) opening a vacuum port to discharge air from a plurality of wells and a plurality of channels, and introducing a vacuum onto the array assembly;
(d) forming an evacuated array by sealing the vacuum ports so that a plurality of wells maintain a vacuum; And
(e) opening the connection opening to introduce the reaction mixture into a plurality of wells through a plurality of channels; including,
The array assembly,
A plurality of wells configured in an array;
A connection opening between the reaction region and the array;
Vacuum port; And
A plurality of channels allowing each well in the array to be in fluid communication with the connection opening and the vacuum port; including,
How to use the array assembly for samples.
The method of claim 14,
Mixing the reaction mixture with one or more reagents disposed in each of a plurality of wells to form a second reaction mixture; And
Performing a second analysis method on the second reaction mixture; further comprising,
How to use the array assembly for samples.
The method of claim 15,
The second reaction mixture is a PCR mixture,
How to use the array assembly for samples.
The method of claim 16,
Thermally cycling the second reaction mixture; further comprising,
How to use the array assembly for samples.
The method of claim 17,
Further comprising: detecting an amplification product in at least one of a plurality of wells
How to use the array assembly for samples.
The method of claim 15,
The sample contains microorganisms, and the one or more reagents contain antibiotics,
How to use the array assembly for samples.
The method of claim 19,
The antibiotic is included at a first concentration in a first well of a plurality of wells, a second concentration is included in a second well of a plurality of wells, and the second concentration is lower than the first concentration. Concentration,
How to use the array assembly for samples.
The method of claim 14,
The step (c) is to provide a vacuum of 2 to 150 mba to the plurality of wells,
How to use the array assembly for samples.
The method of claim 14,
The (c) and (d) steps are performed before the (b) step is completed,
How to use the array assembly for samples.
delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete A sample introduction region, at least one first reaction region in fluid communication with the sample introduction region, a second reaction region in fluid communication with the first reaction region, the second reaction region including a plurality of wells, , The vacuum channel is in fluid communication with the plurality of wells and vacuum ports, the array fill channel extends between the plurality of wells and the first reaction region, and/or the array fill channel is in fluid communication with the plurality of wells and the first reaction region, Preparing a reaction vessel, wherein an open seal is disposed between the first reaction region and the second reaction region;
Performing at least one step of an analysis method on the reaction vessel;
Forming a vacuum partially on the plurality of wells to place at least a portion of the plurality of wells, the vacuum channel, and the array fill channel at a pressure lower than atmospheric pressure;
Sealing a portion of the vacuum channel and forming an exhaust array such that a vacuum is maintained in at least a portion of the plurality of wells and the array fill channel; And
Including, supplying the fluid to the exhaust array by opening an open seal disposed between the first reaction region and the second reaction region so that the fluid flows into the plurality of wells through the array filling channel.
A method of forming an in-situ vacuum in a reaction vessel while performing the analytical method.
The method of claim 52,
At least one step of the analysis method
Introducing a sample into the sample introduction region, inserting the reaction vessel into an apparatus configured to perform the analysis method and partially introduce a vacuum to form an exhaust array, performing one or more reactions in a first reaction region Comprising any one of performing and preparing to supply fluid to the exhaust array,
A method of forming an in-situ vacuum in a reaction vessel while performing the analytical method.
The method of claim 53,
The step of performing one or more reactions in the first reaction zone,
Decomposing a sample to form a decomposition product, separating the decomposition particles from the decomposition product, mixing the decomposition product and silica magnetic beads, collecting the nucleic acid by the silica magnetic beads, and moving the residual decomposition product to a waste chamber , Washing the magnetic beads at least once, moving the washing solution to a waste chamber, eluting nucleic acids from the silica magnetic beads, performing a single or complex first PCR reaction, and the second reaction zone Including any one of the steps of diluting the first PCR reaction product in preparation for performing a second PCR reaction in a plurality of wells of,
A method of forming an in-situ vacuum in a reaction vessel while performing the analytical method.
The method of claim 52,
The reaction vessel further comprises at least one reagent blister in fluid communication with at least one of the sample introduction region, the first reaction region and the second reaction region,
A method of forming an in-situ vacuum in a reaction vessel while performing the analytical method.
The method of claim 55,
The one or more reagent blisters contain dry reagents,
The vacuum forming method further comprises: forming a partial vacuum on the one or more reagent blisters containing the dry reagent.
A method of forming an in-situ vacuum in a reaction vessel while performing the analytical method.
The method of claim 56,
Mixing a fluid with the drying reagent to form a first mixture; further comprising,
A method of forming an in-situ vacuum in a reaction vessel while performing the analytical method.
The method of claim 56,
The at least one reagent blister comprises reagents for sample preparation, nucleic acid recovery, first step PCR and second step PCR,
A method of forming an in-situ vacuum in a reaction vessel while performing the analytical method.
Including a reaction vessel and apparatus,
The reaction vessel includes a sample introduction region, at least one first reaction region in fluid communication with the sample introduction region, and a second reaction region in fluid communication with the first reaction region,
The second reaction zone is
Multiple wells,
A vacuum channel extending between the plurality of wells and vacuum ports and/or in fluid communication with the plurality of wells and vacuum ports,
An array fill channel extending between the plurality of wells and the first reaction region and/or in fluid communication with the plurality of wells and the first reaction region, and
It includes an open seal disposed between the first reaction zone and the second reaction zone,
The apparatus is configured to perform an analytical method using the reaction vessel, and comprises a vacuum system for introducing a partial vacuum to one or more portions of the reaction vessel during performing one or more steps of the analytical method.
system.
The method of claim 59,
At least one portion of the reaction vessel configured to form a partial vacuum is dried such that the partial vacuum is not formed against the partial pressure of water,
system.
The method of claim 60,
The partial vacuum of at least one portion of the reaction vessel configured to form a partial vacuum is 2 to 150 mba,
system.
The method of claim 59,
The at least one portion of the reaction vessel configured to form the partial vacuum comprises at least a portion of a plurality of wells, vacuum channels and array fill channels,
system.
The method of claim 59,
The device comprises a sealing member for applying a seal to preserve a partial vacuum formed in the at least one portion of the reaction vessel.
system.
The method of claim 59,
The reaction vessel further comprises at least one reagent blister in fluid communication with at least one of the sample introduction region, the first reaction region and the second reaction region,
system.
The method of claim 64,
At least one of the one or more reagent blisters comprises a dry reagent,
At least one portion of the reaction vessel configured to form a partial vacuum comprises one or more reagent blisters containing the dry reagent,
system.
The method of claim 59,
The device is a PCR device comprising at least one heater disposed and arranged to thermally cycle at least a portion of the reaction vessel,
system.
The method of claim 59,
The apparatus comprises at least one heater arranged and arranged to control the temperature of at least a portion of the reaction vessel to perform an isothermal reaction,
system.
The method of claim 59,
At least one heater disposed and arranged to control the temperature of at least a portion of the reaction vessel,
One or more actuators for moving fluid to the reaction vessel, and
Comprising one or more seals for controlling the movement of fluid in one or more portions of the reaction vessel,
system.

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